RESUMO
BACKGROUND: Through several not-fully-characterised moonlighting functions, translation elongation factor eEF1A2 is known to provide a fitness boost to cancer cells. Furthermore, eEF1A2 has been demonstrated to confer neoplastic characteristics on preneoplastic, nontumourigenic precursor cells. We have previously shown that eEF1A2 is the target of plitidepsin, a marine drug currently in development for cancer treatment. Herein, we characterised a new signalling pathway through which eEF1A2 promotes tumour cell survival. METHODS: Previously unknown binding partners of eEF1A2 were identified through co-immunoprecipitation, high-performance liquid chromatography-mass spectrometry and proximity ligation assay. Using plitidepsin to release eEF1A2 from those protein complexes, their effects on cancer cell survival were analysed in vitro. RESULTS: We uncovered that double-stranded RNA-activated protein kinase (PKR) is a novel eEF1A2-interacting partner whose pro-apoptotic effect is hindered by the translation factor, most likely through sequestration and inhibition of its kinase activity. Targeting eEF1A2 with plitidepsin releases PKR from the complex, facilitating its activation and triggering a mitogen-activated protein kinase signalling cascade together with a nuclear factor-κB-dependent activation of the extrinsic apoptotic pathway, which lead to tumour cell death. CONCLUSIONS: Through its binding to PKR, eEF1A2 provides a survival boost to cancer cells, constituting an Achilles heel that can be exploited in anticancer therapy.
Assuntos
Sobrevivência Celular , Fator 1 de Elongação de Peptídeos/metabolismo , eIF-2 Quinase/metabolismo , Animais , Células HeLa , Humanos , Camundongos , NF-kappa B/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
BACKGROUND: Vascular supply of tumors is one of the main targets for cancer therapy. Here, we investigated if plocabulin (PM060184), a novel marine-derived microtubule-binding agent, presents antiangiogenic and vascular-disrupting activities. METHODS: The effects of plocabulin on microtubule network and dynamics were studied on HUVEC endothelial cells. We have also studied its effects on capillary tube structures formation or destabilization in three-dimensional collagen matrices. In vivo experiments were performed on different tumor cell lines. RESULTS: In vitro studies show that, at picomolar concentrations, plocabulin inhibits microtubule dynamics in endothelial cells. This subsequently disturbs the microtubule network inducing changes in endothelial cell morphology and causing the collapse of angiogenic vessels, or the suppression of the angiogenic process by inhibiting the migration and invasion abilities of endothelial cells. This rapid collapse of the endothelial tubular network in vitro occurs in a concentration-dependent manner and is observed at concentrations lower than that affecting cell survival. The in vitro findings were confirmed in tumor xenografts where plocabulin treatment induced a large reduction in vascular volume and induction of extensive necrosis in tumors, consistent with antivascular effects. CONCLUSIONS: Altogether, these data suggest that an antivascular mechanism is contributing to the antitumor activities of plocabulin.
Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Policetídeos/farmacologia , Pironas/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Camundongos Nus , Microtúbulos/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/prevenção & controle , Policetídeos/metabolismo , Ligação Proteica , Pironas/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeting microtubules is the most effective wide-spectrum pharmacological strategy in antitumoral chemotherapy, and current research focuses on reducing main drawbacks: neurotoxicity and resistance. PM534 is a novel synthetic compound derived from the Structure-Activity-Relationship study on the natural molecule PM742, isolated from the sponge of the order Lithistida, family Theonellidae, genus Discodermia (du Bocage 1869). PM534 targets the entire colchicine binding domain of tubulin, covering four of the five centers of the pharmacophore model. Its nanomolar affinity and high retention time modulate a strikingly high antitumor activity that efficiently overrides two resistance mechanisms in cells (detoxification pumps and tubulin ßIII isotype overexpression). Furthermore, PM534 induces significant inhibition of tumor growth in mouse xenograft models of human non-small cell lung cancer. Our results present PM534, a highly effective new compound in the preclinical evaluation that is currently in its first human Phase I clinical trial.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Colchicina/metabolismo , Tubulina (Proteína)/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Microtúbulos , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/uso terapêutico , Moduladores de Tubulina/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
The mitochondrial H(+)-ATP synthase is a bottleneck component in the provision of metabolic energy by oxidative phosphorylation. The expression of its catalytic subunit (ß-F1-ATPase) is stringently controlled at post-transcriptional levels during oncogenesis, the cell cycle and in development. Here we show that miR-127-5p targets the 3'UTR of ß-F1-ATPase mRNA (ß-mRNA) significantly reducing its translational efficiency without affecting ß-mRNA abundance. Despite the reduced expression of ß-F1-ATPase in most human carcinomas, we observed no expression of miR-127-5p in different human cancer cell lines, minimizing the potential role of miR-127-5p as a regulator of the bioenergetic activity of mitochondria in cancer. In contrast, miR-127-5p is highly over-expressed in the human fetal liver. Consistent with previous findings in the rat, the expression of ß-F1-ATPase in the human liver also seems to be controlled at post-transcriptional levels during development, what might suggest a role for miR-127-5p in controlling ß-mRNA translation and thus in defining the bioenergetic activity of human liver mitochondria. Moreover, immunolocalization techniques and subcellular fractionation experiments using different antibodies against ß-F1-ATPase reveal that the ectopic expression of ß-F1-ATPase at the cell surface of the hepatocytes and HepG2 cells is negligible or stands for scrutiny.
Assuntos
Regiões 3' não Traduzidas/genética , MicroRNAs/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Biossíntese de Proteínas/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Feto/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/enzimologia , Hepatócitos/ultraestrutura , Humanos , Fígado/embriologia , Fígado/metabolismo , Fígado/ultraestrutura , MicroRNAs/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
Ovarian cancer (OC) is a life-threatening tumor and the deadliest among gynecological cancers in developed countries. First line treatment with a carboplatin/paclitaxel regime is initially effective in the majority of patients, but most advanced OC will recur and develop drug resistance. Therefore, the identification of alternative therapies is needed. In this study, we employed a panel of high-grade serous ovarian cancer (HGSOC) cell lines, in monolayer and three-dimensional cell cultures. We evaluated the effects of a novel tubulin-binding agent, plocabulin, on proliferation, cell cycle, migration and invasion. We have also tested combinations of plocabulin with several drugs currently used in OC in clinical practice. Our results show a potent antitumor activity of plocabulin, inhibiting proliferation, disrupting microtubule network, and decreasing their migration and invasion capabilities. We did not observe any synergistic combination of plocabulin with cisplatin, doxorubicin, gemcitabine or trabectedin. In conclusion, plocabulin has a potent antitumoral effect in HGSOC cell lines that warrants further clinical investigation.
RESUMO
Small-Cell Lung Cancer (SCLC) is an aggressive neuroendocrine malignancy with a poor prognosis. Here, we focus on the neuroendocrine SCLC subtypes, SCLC-A and SCLC-N, whose transcription addiction was driven by ASCL1 and NEUROD1 transcription factors which target E-box motifs to activate up to 40% of total genes, the promoters of which are maintained in a steadily open chromatin environment according to ATAC and H3K27Ac signatures. This leverage is used by the marine agent lurbinectedin, which preferentially targets the CpG islands located downstream of the transcription start site, thus arresting elongating RNAPII and promoting its degradation. This abrogates the expression of ASCL1 and NEUROD1 and of their dependent genes, such as BCL2, INSM1, MYC, and AURKA, which are responsible for relevant SCLC tumorigenic properties such as inhibition of apoptosis and cell survival, as well as for a part of its neuroendocrine features. In summary, we show how the transcription addiction of these cells becomes their Achilles's heel, and how this is effectively exploited by lurbinectedin as a novel SCLC therapeutic endeavor.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Carbolinas , Compostos Heterocíclicos de 4 ou mais Anéis , Neoplasias Pulmonares , Proteínas Repressoras , Carcinoma de Pequenas Células do Pulmão , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carbolinas/farmacologia , Linhagem Celular Tumoral , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismoRESUMO
Melanoma is the cancer with the highest increase in incidence, and transformation of radial growth to vertical growth (i.e., noninvasive to invasive) melanoma is required for invasive disease and metastasis. We have previously shown that p42/p44 MAP kinase is activated in radial growth melanoma, suggesting that further signaling events are required for vertical growth melanoma. The molecular events that accompany this transformation are not well understood. Akt, a signaling molecule downstream of PI3K, was introduced into the radial growth WM35 melanoma in order to test whether Akt overexpression is sufficient to accomplish this transformation. Overexpression of Akt led to upregulation of VEGF, increased production of superoxide ROS, and the switch to a more pronounced glycolytic metabolism. Subcutaneous implantation of WM35 cells overexpressing Akt led to rapidly growing tumors in vivo, while vector control cells did not form tumors. We demonstrated that Akt was associated with malignant transformation of melanoma through at least 2 mechanisms. First, Akt may stabilize cells with extensive mitochondrial DNA mutation, which can generate ROS. Second, Akt can induce expression of the ROS-generating enzyme NOX4. Akt thus serves as a molecular switch that increases angiogenesis and the generation of superoxide, fostering more aggressive tumor behavior. Targeting Akt and ROS may be of therapeutic importance in treatment of advanced melanoma.
Assuntos
Transformação Celular Neoplásica/metabolismo , Melanoma/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Cutâneas/patologia , Animais , Transformação Celular Neoplásica/genética , DNA Mitocondrial/genética , Glicólise , Humanos , Melanoma/irrigação sanguínea , Melanoma/enzimologia , Mitocôndrias/enzimologia , Mutação , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Neovascularização Patológica/enzimologia , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/enzimologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganization and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Policetídeos/farmacologia , Pironas/farmacologia , Moduladores de Tubulina/farmacologia , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Centrossomo/ultraestrutura , Feminino , Interfase , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Prometáfase/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/ultraestrutura , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Trabectedin and Zalypsis are two potent anticancer tetrahydroisoquinoline alkaloids that can form a covalent bond with the amino group of a guanine in selected triplets of DNA duplexes and eventually give rise to double-strand breaks. Using well-defined in vitro and in vivo assays, we show that the resulting DNA adducts stimulate, in a concentration-dependent manner, cleavage by the XPF/ERCC1 nuclease on the strand opposite to that bonded by the drug. They also inhibit RNA synthesis by: (1) preventing binding of transcription factors like Sp1 to DNA, and (2) arresting elongating RNA polymerase II at the same nucleotide position regardless of the strand they are located on. Structural models provide a rationale for these findings and highlight the similarity between this type of DNA modification and an interstrand crosslink.
Assuntos
Antineoplásicos/farmacologia , Quebras de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Dioxóis/farmacologia , RNA Polimerase II/metabolismo , Tetra-Hidroisoquinolinas/farmacologia , Antineoplásicos Alquilantes/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Endonucleases/metabolismo , Humanos , Modelos Moleculares , RNA Polimerase II/genética , Fator de Transcrição Sp1/metabolismo , Trabectedina , Ativação Transcricional/efeitos dos fármacosRESUMO
There is a large body of clinical data documenting that most human carcinomas contain reduced levels of the catalytic subunit of the mitochondrial H+-ATP synthase. In colon and lung cancer this alteration correlates with a poor patient prognosis. Furthermore, recent findings in colon cancer cells indicate that downregulation of the H+-ATP synthase is linked to the resistance of the cells to chemotherapy. However, the mechanism by which the H+-ATP synthase participates in cancer progression is unknown. In this work, we show that inhibitors of the H+-ATP synthase delay staurosporine (STS)-induced cell death in liver cells that are dependent on oxidative phosphorylation for energy provision whereas it has no effect on glycolytic cells. Efficient execution of cell death requires the generation of reactive oxygen species (ROS) controlled by the activity of the H+-ATP synthase in a process that is concurrent with the rapid disorganization of the cellular mitochondrial network. The generation of ROS after STS treatment is highly dependent on the mitochondrial membrane potential and most likely caused by reverse electron flow to Complex I. The generated ROS promote the carbonylation and covalent modification of cellular and mitochondrial proteins. Inhibition of the activity of the H+-ATP synthase blunted ROS production prevented the oxidation of cellular proteins and the modification of mitochondrial proteins delaying the release of cytochrome c and the execution of cell death. The results in this work establish the downregulation of the H+-ATP synthase, and thus of oxidative phosphorylation, as part of the molecular strategy adapted by cancer cells to avoid ROS-mediated cell death. Furthermore, the results provide a mechanistic explanation to understand chemotherapeutic resistance of cancer cells that rely on glycolysis as the main energy provision pathway.
Assuntos
Morte Celular , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Espécies Reativas de Oxigênio , Animais , Carcinoma Hepatocelular/metabolismo , Caspase 3 , Caspases/metabolismo , Núcleo Celular/metabolismo , Eletroforese em Gel Bidimensional , Glicólise , Potenciais da Membrana , Mitocôndrias/patologia , Oxigênio/metabolismo , Ratos , Estaurosporina/farmacologiaRESUMO
Nowadays, we are facing a renaissance of mitochondria in cancer biology. However, our knowledge of the basic cell biology and on the timing and mechanisms that control the biosynthesis of mitochondrial constituents during progression through the cell cycle of mammalian cells remain largely unknown. Herein, we document the in vivo changes on mitochondrial morphology and dynamics that accompany cellular mitosis, and illustrate the following key points of the biogenesis of mitochondria during progression of liver cells through the cycle: (i) the replication of nuclear and mitochondrial genomes is synchronized during cellular proliferation, (ii) the accretion of OXPHOS proteins is asynchronously regulated during proliferation being the synthesis of beta-F1-ATPase and Hsp60 carried out also at G2/M and, (iii) the biosynthesis of cardiolipin is achieved during the S phase, although full development of the mitochondrial membrane potential (DeltaPsim) is attained at G2/M. Furthermore, we demonstrate using reporter constructs that the mechanism regulating the accretion of beta-F1-ATPase during cellular proliferation is controlled at the level of mRNA translation by the 3'UTR of the transcript. The 3'UTR-driven synthesis of the protein at G2/M is essential for conferring to the daughter cells the original phenotype of the parental cell. Our findings suggest that alterations on this process may promote deregulated beta-F1-ATPase expression in human cancer.
Assuntos
Regiões 3' não Traduzidas , Ciclo Celular/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Animais , Ciclo Celular/fisiologia , Linhagem Celular , Células Clonais , Cricetinae , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Los condilomas o verrugas genitales son producidos por el virus del papiloma humano. La infección típicamente ocurre cuando las células basales del huésped se exponen a la infección viral a través de una barrera epitelial dañada, como sucede durante el acto sexual o como producto de otras abrasiones dérmicas menores. Se necesita la presencia de otros factores asociados para producir la enfermedad, como son: promiscuidad sexual, mala nutrición, tabaquismo, multiparidad, depresión del sistema inmunológico y uso prolongado de anticonceptivos orales. Los condilomas acuminados constituyen una de las enfermedades de transmisión sexual más frecuentes y se vinculan, por lo general, con los tipos de cepas 6 y 11. En pocas ocasiones pueden evolucionar como un tumor invasivo con malignidad local, aunque sin metástasis, equivalente a un carcinoma verrucoso: es el síndrome de Buschke-Löwenstein (AU)
Condylomata or genital warts are produced by the human papilloma virus (HPV). The infection occurs typically when the basal cells of the host are exposed to the viral infection due to a damaged epithelial barrier, as happens during the sexual act or in minor skin abrasions. Other associating factors are required to produce the disease, such as: inappropriate sexual behaviour, smoking, malnutrition, multiparity, immune system depression and prolonged use of oral anticonceptives. Condyloma acuminatum is one of the most frequent sexual transmitted diseases and is generally associated with types HPV 6 and 11. On some occasions, it can progress to a local invasive tumor but without metastases, similar to a verrucous carcinoma: it is the Buschke-Löwenstein syndrome (AU)