RESUMO
Germline activating mutations of the protein tyrosine phosphatase SHP2 (encoded by PTPN11), a positive regulator of the RAS signalling pathway, are found in 50% of patients with Noonan syndrome. These patients have an increased risk of developing leukaemia, especially juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative neoplasm (MPN). Previous studies have demonstrated that mutations in Ptpn11 induce a JMML-like MPN through cell-autonomous mechanisms that are dependent on Shp2 catalytic activity. However, the effect of these mutations in the bone marrow microenvironment remains unclear. Here we report that Ptpn11 activating mutations in the mouse bone marrow microenvironment promote the development and progression of MPN through profound detrimental effects on haematopoietic stem cells (HSCs). Ptpn11 mutations in mesenchymal stem/progenitor cells and osteoprogenitors, but not in differentiated osteoblasts or endothelial cells, cause excessive production of the CC chemokine CCL3 (also known as MIP-1α), which recruits monocytes to the area in which HSCs also reside. Consequently, HSCs are hyperactivated by interleukin-1ß and possibly other proinflammatory cytokines produced by monocytes, leading to exacerbated MPN and to donor-cell-derived MPN following stem cell transplantation. Remarkably, administration of CCL3 receptor antagonists effectively reverses MPN development induced by the Ptpn11-mutated bone marrow microenvironment. This study reveals the critical contribution of Ptpn11 mutations in the bone marrow microenvironment to leukaemogenesis and identifies CCL3 as a potential therapeutic target for controlling leukaemic progression in Noonan syndrome and for improving stem cell transplantation therapy in Noonan-syndrome-associated leukaemias.
Assuntos
Transformação Celular Neoplásica/genética , Microambiente Celular/genética , Células-Tronco Hematopoéticas/patologia , Leucemia/genética , Leucemia/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Nicho de Células-Tronco/genética , Animais , Quimiocina CCL3/antagonistas & inibidores , Quimiocina CCL3/metabolismo , Progressão da Doença , Células Endoteliais/citologia , Feminino , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interleucina-1beta/metabolismo , Leucemia Mielomonocítica Juvenil/genética , Leucemia Mielomonocítica Juvenil/metabolismo , Leucemia Mielomonocítica Juvenil/patologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Monócitos/metabolismo , Mutação , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Síndrome de Noonan/patologia , Osteoblastos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transplante de Células-TroncoRESUMO
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.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Antraquinonas/síntese química , Antraquinonas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Células K562 , Estrutura Molecular , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
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.
Assuntos
Sobrevivência Celular/genética , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Ligação Proteica/genética , Estabilidade Proteica , Proteína Supressora de Tumor p53/genética , Ubiquitinação/genética , Regulação para Cima/genéticaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Metilação de DNA , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de Tumor/genética , Sequência de Bases , Linhagem Celular Tumoral , Proteínas Correpressoras , DNA , Primers do DNA , Humanos , Chaperonas Moleculares , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Apoptose , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53 , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Humanos , Animais , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Apoptose/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Relação Dose-Resposta a Droga , Camundongos , Feminino , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Berberina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteínas Correpressoras , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Fluorescência , Humanos , Chaperonas Moleculares , Proteínas Nucleares/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Leucemia Mieloide Aguda , Neoplasias da Próstata , Masculino , Humanos , Animais , Camundongos , Criança , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/farmacologia , Apoptose , Linhagem Celular Tumoral , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Regiões 5' não Traduzidas , Antineoplásicos Fitogênicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Biossíntese de Proteínas , Fator 1 Associado a Receptor de TNF/genética , Vincristina/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Proteína de Ligação a Regiões Ricas em Polipirimidinas/análise , Biossíntese de Proteínas/efeitos dos fármacos , Deleção de Sequência , Fator 1 Associado a Receptor de TNF/biossíntese , Células Tumorais CultivadasRESUMO
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.
RESUMO
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.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Quinolinas/farmacologia , Sulfonamidas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Quinolinas/síntese química , Quinolinas/metabolismo , Ratos Sprague-Dawley , Sulfonamidas/síntese química , Sulfonamidas/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Transmembrane TNF-alpha (tmTNF-alpha) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-alpha (tmTNF-alpha) at high level was resistant to soluble TNF-alpha (sTNF-alpha)-induced cytotoxicity, accompanied by constitutive NF-kappaB activation. In contrast, MCF-7 cells expressing tmTNF-alpha at very low level were sensitive to sTNF-alpha-induced cell death and had no detectable NF-kappaB activation. Consistently, siRNA-mediated tmTNF-alpha knockdown blocked NF-kappaB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-alpha, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-alpha containing LS constitutively activated NF-kappaB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-alpha lacking the cytoplasmic segment of LS neither activated NF-kappaB nor affected the sensitivity. However, NF-kappaB inhibitor PDTC suppressed NF-kappaB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-kappaB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)(2)-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-kappaB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-kappaB and resistance to sTNF-alpha-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-alpha-induced resistance in the breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismo , Western Blotting , Morte Celular/fisiologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Microscopia Confocal , NF-kappa B/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Transfecção , Fator de Necrose Tumoral alfa/genéticaRESUMO
Interestingly, some lymphoma cells, expressing high levels of transmembrane (tm)TNF-alpha, are resistant to secretory (s)TNF-alpha-induced necrosis but sensitive to tmTNF-alpha-mediated apoptosis. As tmTNF-alpha mediates "forward" as well as "reverse" signaling, we hypothesize that a balanced signaling between forward and reverse directions may play a critical role in determining the fate of cells bearing tmTNF-alpha. Using Raji cells as a model, we first added exogenous tmTNF-alpha on fixed, transfected NIH3T3 cells onto Raji cells to examine tmTNF-alpha forward signaling and its effects, showing that constitutive NF-kappaB activity and cellular inhibitor-of-apoptosis protein 1 transcription were down-regulated, paralleled with Raji cell death. As Raji cells express tmTNF-alpha, an inhibition of their tmTNF-alpha expression by antisense oligonucleotide caused down-regulation of NF-kappaB activity. Conversely, increasing tmTNF-alpha expression by suppressing expression of TNF-alpha-converting enzyme that cleaves tmTNF-alpha led to an enhanced activation of NF-kappaB, indicating that tmTNF-alpha, but not sTNF-alpha, contributes to constitutive NF-kappaB activation. We next transfected Raji cells with a mutant tmTNF-alpha lacking the intracellular domain to competitively suppress reverse signaling via tmTNF-alpha; as expected, constitutive NF-kappaB activity was decreased. In contrast, treating Raji cells with sTNFR2 to stimulate reverse signaling via tmTNF-alpha enhanced NF-kappaB activation. We conclude that tmTNF-alpha, when highly expressed on tumor cells and acting as a receptor, promotes NF-kappaB activation through reverse signaling, which is helpful to maintain tumor cell survival. On the contrary, tmTNF-alpha, when acting as a ligand, inhibits NF-kappaB activity through forward signaling, which is inclined to induce tumor cell death.
Assuntos
Apoptose/efeitos dos fármacos , Linfoma de Burkitt/patologia , Membrana Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Luciferases/metabolismo , NF-kappa B/genética , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genéticaRESUMO
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.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Imidazóis/farmacologia , Leucemia Linfoide/tratamento farmacológico , Leucemia Linfoide/metabolismo , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Doença Aguda , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose , Morte Celular , Cromonas/farmacologia , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Morfolinas/farmacologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , RNA Interferente Pequeno , Survivina , Transfecção , Células Tumorais CultivadasRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Proliferação de Células , DNA Topoisomerases Tipo II , Regulação Neoplásica da Expressão Gênica , Neoplasias , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Proto-Oncogênicas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Apoptose/fisiologia , Doxorrubicina/farmacologia , Fator VIIa/metabolismo , Genes bcl-2/fisiologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Tromboplastina/metabolismo , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT5/metabolismo , Regulação para CimaRESUMO
The effects of tissue factor (TF) on doxorubicin-induced apoptosis in human neuroblastoma were investigated. The expression of TF was examined by Western blotting. TFsiRNA-pSUPER plasmid was constructed by inserting specific 19-nt silencing sequence targeting TF gene into pSUPER vector. Transfection of TFsiRNA-pSUPER was performed using lipofectamine2000. The cytotoxicity of doxorubicin was determined by WST assay. The activation of Caspase-3 and PARP induced by doxorubicin was tested by Western blotting. The apoptotic cells were stained by Hochest33342 and counted under fluorescence inverted microscope. It was found that human neuroblastoma cell line SK-N-MC expressed high level of TF. Knockdown of the TF expression was achieved by transfection of TFsiRNA-pSUPER on SK-N-MC cells in a dose-dependent manner. Inhibition of TF significantly decreased the viability of transfected SK-N-MC cells treated with different concentrations of doxorubicin. Cleavage of Caspase-3 and PARP was enhanced in transfected SK-N-MC cells with down-regulation of TF. TFsiRNA treatment significantly increased the number of apoptotic cells in transfected SK-N-MC cells as compared with those control cells (P<0.05) when these cells were exposed to 1 mug/mL doxorubicin for 8 h. These results suggested that knockdown of the TF expression by specific siRNA vector could increase the cytotoxicity of doxorubicin and enhance doxorubicin-induced apoptosis in human neuroblastoma cells. Over-expression of TF might contribute to chemotherapy resistance in human neuroblastoma and its progression, at lest in part, by regulating doxorubicin-induced apoptosis.
Assuntos
Apoptose , Regulação para Baixo , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Neuroblastoma/metabolismo , RNA Interferente Pequeno , Tromboplastina/biossíntese , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Caspase 3/metabolismo , Humanos , Concentração Inibidora 50 , Modelos Biológicos , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de TempoRESUMO
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.
Assuntos
Antígenos CD34 , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/biossíntese , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Células-Tronco Hematopoéticas/citologia , Humanos , Proteínas Inibidoras de Apoptose , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/fisiologia , Inibidores de Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transdução de Sinais/fisiologia , SurvivinaRESUMO
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.
Assuntos
Antraquinonas/uso terapêutico , Neoplasias/genética , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Animais , Antraquinonas/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias/tratamento farmacológicoRESUMO
BACKGROUND: The overexpression of eukaryotic translation initiation factor 4E (eIF4E), a key regulator of protein synthesis, is involved in the malignant progression of human breast cancer. This study investigates the relationship between eIF4E and angiogenesis, as well as their prognostic impact in patients with human breast cancer. METHODS: Immunohistochemical staining was used to determine protein expression of eIF4E, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and CD105 in a set of 122 formalin-fixed, paraffin-embedded primary breast cancer tissues. Expression of eIF4E in positive cells was characterized by cytoplasmic staining. Evaluation of VEGF and IL-8 in the same tissue established the angiogenic profiles, while CD105 was used as an indicator of microvessel density (MVD). RESULTS: A significant relationship was found between the level of eIF4E expression and histological grade (P = 0.016). VEGF, IL-8, and MVD were closely related to tumor grade (P = 0.003, P = 0.022, and P < 0.001, respectively) and clinical stage (P = 0.007, P = 0.048, and P < 0.001, respectively). Expression of eIF4E was also significantly correlated with VEGF (P = 0.007), IL-8 (P = 0.007), and MVD (P = 0.006). Patients overexpressing eIF4E had significantly worse overall (P = 0.01) and disease-free survival (P = 0.006). When eIF4E, histological grade, tumor stage, ER, PR, Her-2 status and the levels of VEGF, IL-8, MVD were included in a multivariate Cox regression analysis, eIF4E emerged as an independent prognostic factor for breast cancer (P = 0.001), along with stage (P = 0.005), node status (P = 0.046), and MVD (P = 0.004). CONCLUSION: These results suggest that higher eIF4E expression correlates with both angiogenesis and vascular invasion of cancer cells, and could therefore serve as a useful histological predictor for less favorable outcome in breast cancer patients, as well as represent a potential therapeutic target.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/irrigação sanguínea , Fator de Iniciação 4E em Eucariotos/fisiologia , Interleucina-8/biossíntese , Proteínas de Neoplasias/fisiologia , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Adulto , Idoso , Antígenos CD/biossíntese , Antígenos CD/genética , Neoplasias da Mama/química , Neoplasias da Mama/mortalidade , Progressão da Doença , Intervalo Livre de Doença , Endoglina , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Interleucina-8/genética , Tábuas de Vida , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Prognóstico , Modelos de Riscos Proporcionais , Biossíntese de Proteínas , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Análise de Sobrevida , Taxa de Sobrevida , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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.