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1.
Int J Cancer ; 138(2): 497-506, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26260582

RESUMO

Since polo-like kinase 1 (PLK1) is highly expressed in Ewing sarcoma (ES), we evaluated the therapeutic potential of the PLK1 inhibitor BI 6727. Here, we identify a synergistic induction of apoptosis by BI 6727 and several microtubule-interfering drugs in ES cells, including vincristine (VCR), vinblastine (VBL), vinorelbine (VNR) and eribulin. Synergistic drug interaction is confirmed by calculation of combination index (CI). Also, BI 6727 and VCR act in concert to reduce long-term clonogenic survival. Mechanistically, BI 6727/VCR co-treatment cooperates to trigger mitotic arrest, phosphorylation of BCL-2 and BCL-XL and downregulation of MCL-1. This inactivation of anti-apoptotic BCL-2 family proteins in turn promotes activation of BAX and BAK, activation of caspase-9 and -3 and caspase-dependent apoptosis. Overexpression of BCL-2 or simultaneous knockdown of BAX and BAK significantly rescue BI 6727/VCR-induced apoptosis, indicating that engagement of the mitochondrial pathway is critical for BI 6727/VCR-mediated apoptosis. The clinical relevance of PLK1 inhibitor-based combination therapies is underscored by the fact that BI 6727 is currently evaluated in phase I clinical trials in childhood cancer. In conclusion, PLK1 inhibitors such as BI 6727 may provide a new strategy to chemosensitize ES.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/patologia , Sarcoma de Ewing/patologia , Western Blotting , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/administração & dosagem , Interferência de RNA , Moduladores de Tubulina/administração & dosagem , Quinase 1 Polo-Like
2.
J Biol Chem ; 288(49): 35287-96, 2013 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-24133218

RESUMO

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.


Assuntos
Compostos de Bifenilo/administração & dosagem , Morfolinas/administração & dosagem , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Nitrofenóis/administração & dosagem , Rabdomiossarcoma/tratamento farmacológico , Sulfonamidas/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Inibidores de Fosfoinositídeo-3 Quinase , Piperazinas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos
3.
Int J Cancer ; 132(11): 2682-93, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23151917

RESUMO

On the basis of our previous identification of aberrant phosphatidylinositol-3-kinase (PI3K)/Akt signaling as a novel poor prognostic factor in neuroblastoma, we evaluated the dual PI3K/mTOR inhibitor BEZ235 in the present study. Here, BEZ235 acts in concert with the lysosomotropic agent chloroquine (CQ) to trigger apoptosis in neuroblastoma cells in a synergistic manner, as calculated by combination index (CI < 0.5). Surprisingly, inhibition of BEZ235-induced autophagy is unlikely the primary mechanism of this synergism as reported in other cancers, since neither inhibition of autophagosome formation by knockdown of Atg7 or Atg5 nor disruption of the autophagic flux by Bafilomycin A1 (BafA1) enhance BEZ235-induced apoptosis. BEZ235 stimulates enlargement of the lysosomal compartment and generation of reactive oxygen species (ROS), while CQ promotes lysosomal membrane permeabilization (LMP). In combination, BEZ235 and CQ cooperate to trigger LMP, Bax activation, loss of mitochondrial membrane potential (MMP) and caspase-dependent apoptosis. Lysosome-mediated apoptosis occurs in a ROS-dependent manner, as ROS scavengers significantly reduce BEZ235/CQ-induced loss of MMP, LMP and apoptosis. There is a mitochondrial-lysosomal cross-talk, since lysosomal enzyme inhibitors significantly decrease BEZ235- and CQ-induced drop of MMP and apoptosis. In conclusion, BEZ235 and CQ act in concert to trigger LMP and lysosome-mediated apoptosis via a mitochondrial-lysosomal cross-talk. These findings have important implications for the rational development of PI3K/mTOR inhibitor-based combination therapies.


Assuntos
Apoptose/efeitos dos fármacos , Cloroquina/farmacologia , Imidazóis/farmacologia , Lisossomos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neuroblastoma/patologia , Quinolinas/farmacologia , Antimaláricos/farmacologia , Autofagia , Western Blotting , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Humanos , Imunoprecipitação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2/metabolismo
4.
Cancer Lett ; 440-441: 126-134, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30312727

RESUMO

Multidrug resistance (MDR) in cancer patients undergoing chemotherapy is preventing effective treatment of multiple cancer types including pediatric tumors. Resistance to chemotherapeutic drugs in cancer cells is frequently associated with high expression of p-glycoprotein, a transporter in the plasma membrane that can mediate cellular drug export. Here, we generated pediatric cancer cells with acquired resistance to the chemotherapeutic drug vincristine (VCR). In these cells, acquired resistance is associated with increased expression of p-glycoprotein. VCR-resistant cells display an MDR phenotype and have acquired resistance to multiple other chemotherapeutic drugs including doxorubicin (DOXO) and etoposide (ETO). Notably, we discovered that these cells also display cross-resistance with several Smac mimetics, a novel class of experimental cancer therapeutics designed to induce apoptosis by inhibiting Inhibitor of Apoptosis (IAP) proteins. Resistance to Smac mimetics is reversible in the presence of p-glycoprotein inhibitors, highlighting Smac mimetics as novel substrates for p-glycoprotein. The identification of Smac mimetics as substrates for p-glycoproteins may influence the design of future clinical trials to prevent usage of Smac mimetics in the context of MDR or, alternatively, combine Smac mimetics with p-glycoprotein inhibitors to maximize their efficiency.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Materiais Biomiméticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Neuroblastoma/tratamento farmacológico , Rabdomiossarcoma/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Humanos , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Oligopeptídeos/farmacologia , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Tiazóis/farmacologia , Regulação para Cima , Vincristina/farmacologia
5.
Cell Death Differ ; 26(10): 2046-2060, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30742091

RESUMO

Resistance to apoptosis is a hallmark of cancer and deregulation of apoptosis often leads to chemoresistance. Therefore, new approaches to target apoptosis-resistant cancer cells are crucial for the development of directed cancer therapies. In the present study, we investigated the effect of cell cycle regulators on interferon (IFN)-induced necroptosis as an alternative cell death mechanism to overcome apoptosis resistance. Here, we report a novel combination treatment of IFNs with cell cycle arrest-inducing compounds that induce necroptosis in apoptosis-resistant cancer cells and elucidate the underlying molecular mechanisms. Combination treatment of IFNs (i.e. IFNß) with inhibitors of the cell cycle (e.g. vinorelbine (VNR), nocodazole (Noc), polo-like kinase-1 (Plk-1) inhibitor BI 6727) co-operate to induce necroptotic cell death upon caspase inactivation. The mode of cell death was confirmed by pharmacological inhibition and siRNA-mediated downregulation of the key necroptotic factors receptor-interacting protein (RIP) kinase 3 (RIP3) and mixed-lineage kinase-like (MLKL) in various cell lines. Mechanistically, we show that necroptosis upon VNR/IFNß/zVAD.fmk treatment is RIP1-independent but relies on IFNß-induced gene expression of Z-DNA-binding protein 1 (ZBP1) as shown by quantitative RT-PCR and genetic knockdown experiments. Interestingly, we find that RIP3 is phosphorylated in response to compounds that trigger mitotic arrest, even in the absence of IFNß signaling and necroptosis induction. Together, the identification of a novel combination treatment that triggers necroptosis has implications for the development of molecular-targeted therapies to circumvent apoptosis resistance and point to an underestimated role of cell cycle regulation in cell death signaling.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Mitose/fisiologia , Necroptose/genética , Animais , Humanos , Interferons/metabolismo , Camundongos , Transfecção
6.
Cancer Res ; 78(8): 2000-2013, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29437705

RESUMO

Sequencing studies have revealed recurrent mutations in the RAS pathway in rhabdomyosarcoma (RMS). However, RAS effector pathways in RMS are poorly defined. Here, we report that coinhibition of NRAS or MEK plus PI3Kα triggers widespread apoptosis in NRAS-mutated RMS cells. Subtoxic concentrations of the MEK inhibitor MEK162 and the PI3Kα-specific inhibitor BYL719 synergized to trigger apoptosis in NRAS-mutated RMS cells in vitro and in vivoNRAS- or HRAS-mutated cell lines were more vulnerable to MEK162/BYL719 cotreatment than RAS wild-type cell lines, and MEK162/BYL719 cotreatment was more effective to trigger apoptosis in NRAS-mutated than RAS wild-type RMS tumors in vivo We identified BCL-2-modifying factor (BMF) as an inhibitory target of oncogenic NRAS, with either NRAS silencing or MEK inhibition upregulating BMF mRNA and protein levels, which BYL719 further increased. BMF silencing ablated MEK162/BYL719-induced apoptosis. Mechanistic investigations implicated a proapoptotic rebalancing of BCL-2 family members and suppression of cap-dependent translation in apoptotic sensitivity upon MEK162/BYL719 cotreatment. Our results offer a rationale for combining MEK- and PI3Kα-specific inhibitors in clinical treatment of RAS-mutated RMS.Significance: These findings offer a mechanistic rationale for combining MEK- and PI3Kα-specific inhibitors in the clinical treatment of RAS-mutated forms of often untreatable rhabdomyosarcomas. Cancer Res; 78(8); 2000-13. ©2018 AACR.


Assuntos
Apoptose , Genes ras , MAP Quinase Quinase Quinases/antagonistas & inibidores , Mitocôndrias/metabolismo , Mutação , Proteínas Nucleares/antagonistas & inibidores , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Fatores de Transcrição/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Benzimidazóis/administração & dosagem , Benzimidazóis/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Humanos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Capuzes de RNA , Tiazóis/administração & dosagem , Tiazóis/farmacologia
7.
Oncotarget ; 8(32): 52445-52456, 2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-28881742

RESUMO

In this study, we investigated the molecular mechanisms of eribulin-induced cell death and its therapeutic potential in combination with the PLK1 inhibitor BI 6727 in Ewing sarcoma (ES). Here, we show that eribulin triggers cell death in a dose-dependent manner in a panel of ES cell lines. In addition, eribulin at subtoxic, low nanomolar concentrations acts in concert with BI 6727 to induce cell death and to suppress long-term clonogenic survival. Mechanistic studies reveal that eribulin monotherapy at cytotoxic concentrations and co-treatment with eribulin at subtoxic concentrations together with BI 6727 arrest cells in the M phase of the cell cycle prior to the onset of cell death. This mitotic arrest is followed by increased phosphorylation of BCL-2 and BCL-xL as well as downregulation of MCL-1, suggesting inactivation of these antiapoptotic BCL-2 family proteins. Consistently, eribulin monotherapy and eribulin/BI 6727 co-treatment trigger activation of BAX, a key proapoptotic BCL-2 family protein, and increase proteolytic activation of caspase-9 and -3. Importantly, overexpression of BCL-2 or addition of the broad-range caspase inhibitor zVAD.fmk significantly rescue eribulin- as well as eribulin/BI 6727-induced cell death. Together, these findings demonstrate that eribulin induces cell death via the intrinsic pathway of apoptosis in ES cells, both alone at cytotoxic concentrations and in combination with BI 6727 at subtoxic concentrations. Thus, our study highlights the therapeutic potential of eribulin for the treatment of ES alone or in rational combination therapies.

8.
Oncotarget ; 7(8): 8700-11, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26046302

RESUMO

High polo-like kinase 1 (PLK1) expression has been linked to poor outcome in neuroblastoma (NB), indicating that it represents a relevant therapeutic target in this malignancy. Here, we identify a synergistic induction of apoptosis by the PLK1 inhibitor BI 2536 and vinca alkaloids in NB cells. Synergistic drug interaction of BI 2536 together with vincristine (VCR), vinblastine (VBL) or vinorelbine (VNR) is confirmed by calculation of combination index (CI). Also, BI 2536 and VCR act in concert to reduce long-term clonogenic survival. Importantly, BI 2536 significantly enhances the antitumor activity of VCR in an in vivo model of NB. Mechanistically, BI 2536/VCR co-treatment triggers prolonged mitotic arrest, which is necessary for BI 2536/VCR-mediated apoptosis, since pharmacological inhibition of mitotic arrest by the CDK1 inhibitor RO-3306 significantly reduces cell death. Prolonged mitotic arrest leads to phosphorylation-mediated inactivation of BCL-2 and BCL-XL as well as downregulation of MCL-1, since inhibition of mitotic arrest by RO-3306 also prevents phosphorylation of BCL-2 and BCL-XL and MCL-1 downregulation. This inactivation of antiapoptotic BCL-2 proteins promotes activation of BAX and BAK, cleavage of caspase-9 and -3 and caspase-dependent apoptosis. Engagement of the mitochondrial pathway of apoptosis is critically required for BI 2536/VCR-induced apoptosis, since ectopic expression of a non-degradable MCL-1 phospho-mutant, BCL-2 overexpression or BAK knockdown significantly reduce BI 2536/VCR-mediated apoptosis. Thus, PLK1 inhibitors may open new perspectives for chemosensitization of NB.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Alcaloides de Vinca/farmacologia , Antineoplásicos/farmacologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Neuroblastoma/enzimologia , Células Tumorais Cultivadas , Quinase 1 Polo-Like
9.
Cancer Lett ; 381(2): 287-95, 2016 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-27521572

RESUMO

The prognosis of metastatic or relapsed rhabdomyosarcoma (RMS) is poor, highlighting the need of new treatment options. In the present study, we evaluated the in vitro efficacy of arsenic trioxide (ATO) in RMS, a FDA-approved drug used in pediatric leukemia. Here, we report that ATO exerts antitumor activity against RMS cells both as single agent and in combination with microtubule-targeting drugs. Monotherapy with ATO reduces cell viability, triggers apoptosis and suppresses clonogenic survival of RMS cells, at least in part, by transcriptional induction of the proapoptotic BH3-only protein Noxa. siRNA-mediated knockdown of Noxa significantly rescues ATO-mediated cell death, demonstrating that Noxa is required for cell death. Also, ATO suppresses endogenous Hedgehog (Hh) signaling, as it significantly reduces Gli1 transcriptional activity and expression levels of several Hh target genes. Furthermore, we identify synergistic induction of apoptosis by ATO together with several antimicrotubule agents including vincristine (VCR), vinblastine and eribulin. The addition of the broad-range caspase inhibitor zVAD.fmk or overexpression of the antiapoptotic protein Bcl-2 significantly reduce ATO/VCR-induced cell death, indicating that the ATO/VCR combination triggers caspase-dependent apoptosis via the mitochondrial pathway. In summary, ATO exerts antitumor activity against RMS, especially in combination with antimicrotubule drugs. These findings have important implications for the development of novel therapeutic strategies for RMS.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Óxidos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Rabdomiossarcoma/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Trióxido de Arsênio , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Furanos/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Cetonas/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Regulação para Cima , Vimblastina/farmacologia , Vincristina/farmacologia , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
10.
Cancer Lett ; 360(1): 1-9, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-25637161

RESUMO

Aberrant activation of the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway has been reported for rhabdomyosarcoma (RMS) and is implicated in survival of tumor cells as well as therapeutic resistance. In the present study, we searched for combination therapies with the dual PI3K/mTOR inhibitor NVP-BEZ235 (BEZ235) in RMS. Here, we identify a synthetic lethal interaction of BEZ235 together with the lysosomotropic agent chloroquine (CQ), which is effective against embryonal rhabdomyosarcoma (ERMS). BEZ235 and CQ at subtoxic concentrations synergize to induce apoptosis in ERMS cells, as confirmed by calculation of combination index (CI). BEZ235 and CQ cooperate to activate caspase-9, -3 and -8, which is crucial for apoptosis induction given that the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) blocks BEZ235/CQ-induced apoptosis. Additionally, pharmacological inhibition of lysosomal enzymes significantly reduces BEZ235/CQ-induced apoptosis, indicating concomitant activation of the lysosomal compartment. Importantly, BEZ235/CQ-induced apoptosis is significantly inhibited by antioxidants, implying that increased oxidative stress contributes to BEZ235/CQ-induced cell death. Importantly, our molecular studies reveal that BEZ235/CQ-induced apoptosis is mediated by cooperative downregulation of the antiapoptotic BCL-2 family protein MCL-1, since stabilization of MCL-1 by expression of a non-degradable MCL-1 phospho-defective mutant significantly decreases BEZ235/CQ-induced apoptosis. Also, overexpression of antiapoptotic BCL-2 leads to a significant reduction of BEZ235/CQ-induced apoptosis, emphasizing that an intact mitochondrial pathway of apoptosis is required for BEZ235/CQ-induced cell death. This identification of a synthetic lethality of BEZ235 and CQ has important implications for the development of molecular targeted therapies for RMS.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Rabdomiossarcoma Embrionário/enzimologia , Rabdomiossarcoma Embrionário/patologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Caspases/metabolismo , Linhagem Celular Tumoral , Cloroquina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática , Humanos , Imidazóis/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Terapia de Alvo Molecular , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quinolinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Rabdomiossarcoma Embrionário/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Transfecção
11.
Cancer Lett ; 365(1): 37-46, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25917079

RESUMO

Eribulin, a novel microtubule-interfering drug, was recently shown to exhibit high antitumor activity in vivo against various pediatric cancers. Here, we identify a novel synthetic lethal interaction of Eribulin together with Polo-like kinase 1 (PLK1) inhibitors against rhabdomyosarcoma (RMS) in vitro and in vivo. Eribulin and the PLK1 inhibitor BI 2536 at subtoxic concentrations synergize to induce apoptosis in RMS cells as confirmed by calculation of combination index (CI). Also, Eribulin/BI 2536 co-treatment is significantly more effective than monotherapy to reduce cell viability and inhibit colony formation of RMS cells. Similarly, Eribulin and BI 2536 act in concert to trigger apoptosis in a primary, patient-derived ARMS culture, underscoring the clinical relevance of this combination. Importantly, Eribulin and BI 2536 cooperate to suppress tumor growth in an in vivo model of RMS. On molecular grounds, Eribulin/BI 2536 co-treatment causes profound mitotic arrest, which is critically required for synergism, since inhibition of mitotic arrest by CDK1 inhibitor RO-3306 abolishes Eribulin/BI 2536-mediated apoptosis. Eribulin and BI 2536 cooperate to activate caspase-9, -3 and -8, which is necessary for apoptosis induction, since the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) reduces Eribulin/BI 2536-induced apoptosis significantly, yet partially. Intriguingly, knockdown of endonuclease G (ENDOG) also significantly inhibits Eribulin/BI 2536-triggered apoptosis, demonstrating the involvement of both caspase-dependent and -independent effector pathways. Synergistic induction of apoptosis is similarly found for Eribulin/BI 2536 co-treatment in neuroblastoma cells and for the combination of vincristine (another antimicrotubule chemotherapeutic) with Poloxin (another PLK1 inhibitor), thus pointing to a broader significance of this concomitant microtubule- and PLK1-targeting strategy for pediatric oncology. In conclusion, the identification of a novel synthetic lethality by dual targeting of mitosis using microtubule-interfering and PLK1-targeted drugs, i.e. Eribulin and BI 2536, has important implications for the development of more effective treatment strategies for RMS.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Rabdomiossarcoma/enzimologia , Rabdomiossarcoma/patologia , Animais , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Furanos/farmacologia , Humanos , Cetonas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/farmacologia , Interferência de RNA , Rabdomiossarcoma/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Moduladores de Tubulina/farmacologia , Carga Tumoral/efeitos dos fármacos , Quinase 1 Polo-Like
12.
Oncotarget ; 6(39): 41522-34, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26575016

RESUMO

We explored the potential of Smac mimetics, which antagonize Inhibitor of Apoptosis (IAP) proteins, for chemosensitization of neuroblastoma (NB). Here, we report that Smac mimetics, e.g. BV6, prime NB cells for chemotherapeutics including the topoisomerase II inhibitor doxorubicin (DOX) and vinca alkaloids such as Vincristine (VCR), Vinblastine (VBL) and Vinorelbine (VNR). Additionally, BV6 acts in concert with DOX or VCR to suppress long-term clonogenic growth. While BV6 causes rapid downregulation of cellular IAP (cIAP)1 protein and nuclear factor-kappaB (NF-κB) activation, DOX/BV6- or VCR/BV6-induced apoptosis occurs independently of NF-κB or TNFα signaling, since overexpression of dominant-negative IκBα superrepressor or the Tumor Necrosis Factor (TNF)α-blocking antibody Enbrel fail to block cell death. Mechanistic studies reveal that Receptor-interacting protein (RIP)1 is required for DOX/BV6-, but not for VCR/BV6-induced apoptosis, since transient or stable knockdown of RIP1 or the pharmacological RIP1 inhibitor necrostatin-1 significantly reduce apoptosis. By comparison, VCR/BV6-mediated apoptosis critically depends on the mitochondrial pathway. VCR/BV6 cotreatment causes phosphorylation of BCL-2 during mitotic arrest, enhanced activation of BAX and BAK and loss of mitochondrial membrane potential (MMP). Additionally, overexpression of BCL-2 profoundly suppresses VCR/BV6-induced apoptosis. Thus, BV6 sensitizes NB cells to chemotherapy-induced apoptosis via distinct initial signaling mechanisms depending on the chemotherapeutic drug. These findings provide novel mechanistic insights into Smac mimetic-mediated chemosensitization of NB.


Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Materiais Biomiméticos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Neuroblastoma/tratamento farmacológico , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Oligopeptídeos/farmacologia , Proteínas de Ligação a RNA/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Antígeno CD47/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Ativação Enzimática , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Inibidores da Topoisomerase II/farmacologia , Transfecção , Vincristina/farmacologia
13.
Curr Pharm Biotechnol ; 13(11): 2248-58, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21605069

RESUMO

The tumor necrosis factor (TNF) gene is an immediate early gene, rapidly transcribed in a variety of cell types following exposure to a broad range of pathogens and signals of inflammation and stress. Regulation of TNF gene expression at the transcriptional level is cell type- and stimulus-specific, involving epigenetic mechanisms or miRNAs. A better knowledge of the molecular mechanisms that control TNF gene regulation and TNF signalling will provide deeper understanding of the initiation and development of apoptotic and inflammatory processes triggered by TNF cytokine in the gut. The described efforts to embed TNF in clinical treatment regiments reflect its attractive effectiveness in killing tumor cells. Whether the described strategies will achieve the success of incorporating TNF in lower gastrointestinal tract therapy for inflammatory diseases and cancer remains to be determined.


Assuntos
Apoptose/fisiologia , Trato Gastrointestinal Inferior/metabolismo , Fatores de Necrose Tumoral/metabolismo , Animais , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/radioterapia , Transdução de Sinais
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