RESUMEN
Nitric oxide is a pleiotropic free radical produced by three nitric oxide synthases (NOS1-3), of which inducible NOS2 is involved in tumor initiation and progression. In this study, RNA-seq, ChIP-seq and qRT-PCR experiments combined with bioinformatic analyses showed that NRF2 is a repressor of NOS2 gene by maintaining a distal enhancer located 22 kb downstream of TSS in an inactive state. Deletion of NRF2 leads to activation of the enhancer, which exerts a pioneering function before it is fully activated. Specifically, NRF2 controls the expression of NOS2 in response to intracellular oxidative stress and extracellular oxygen pressure. We found that abrogation of NOS2 expression by siRNAs partially reduced the ability of WT Panc-1 cells to form 3D spheroids, but strongly reduced the formation of 3D spheroids by NRF2-depleted Panc-1 cells. Mechanistically, this effect correlates with the finding that NOS2 and nitric oxide stimulate epithelial-to-mesenchymal transition in NRF2-depleted Panc-1 and MIA PaCa-2 cells. We also found that knockdown of NOS2 leads to blockade of 3D matrigel invasion of NRF2-depleted PDAC cells, demonstrating that a short-circuit in the reciprocal regulation of NOS2 and NRF2 attenuates the malignancy of PDAC cells. In summary, we show for the first time that: (i) NRF2 is a suppressor of NOS2 in pancreatic cancer cells; (ii) NRF2 binds to and inactivates an enhancer located 22 kb downstream of TSS of the NOS2 gene; (iii) activation of NOS2 requires suppression of NRF2; (iv) NOS2 is required for NRF2-depleted Panc-1 cells to maintain their malignancy and invasiveness.
Asunto(s)
Factor 2 Relacionado con NF-E2 , Neoplasias Pancreáticas , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
In pancreatic ductal adenocarcinomas (PDAC), the KRASG12D-NRF2 axis controls cellular functions such as redox homeostasis and metabolism. Disruption of this axis through suppression of NRF2 leads to profound reprogramming of metabolism. Unbiased transcriptome and metabolome analyses showed that PDAC cells with disrupted KRASG12D-NRF2 signaling (NRF2-/- cells) shift from aerobic glycolysis to metabolic pathways fed by amino acids. Metabolome, RNA-seq and qRT-PCR analyses revealed a blockade of the urea cycle, making NRF2-/- cells dependent on exogenous arginine for survival. Arginine is channeled into anabolic pathways, including the synthesis of phosphocreatine, which generates an energy buffer essential for cell growth. A similar switch was observed in tumor clones that had survived FOLFIRINOX therapy or blockade of KRAS signaling. Inhibition of the creatine pathway with cyclocreatine reduced both ATP and invasion rate in 3D spheroids from NRF2-deficient PDAC cells. Our study provides basis for the rational development of combination therapies for pancreatic cancer.
RESUMEN
Photodynamic therapy (PDT) is a therapeutic modality based on the simultaneous action of three elements: photosensitizer, light and oxygen. This triad generates singlet oxygen and reactive oxygen species that can reduce the mass of a tumor. PDT is also able to stimulate iNOS, the enzyme that generates nitric oxide (NO). The role of NO in PDT-treated cancer cells has been investigated in several studies. They showed that low iNOS/NO levels stimulate signaling pathways that promote tumor survival, while high iNOS/NO levels arrest tumor growth. There is increasing evidence that ROS/RNS control both proliferation and migration of cells in the vicinity of PDT-treated tumor cells (so-called bystander cells). In this work, we addressed the question of how NO, which is generated by weak PDT, affects bystander cells. We used a conditioned medium: medium of PDT-treated tumor cells containing the stressors produced by the cells was added to untreated cells mimicking the neighboring bystander cells to investigate whether the conditioned medium affects cell proliferation. We found that low-level NO in prostate cancer cells affects the bystander tumor cells in a manner that depends on their malignancy grade.
Asunto(s)
Fotoquimioterapia , Neoplasias de la Próstata , Efecto Espectador , Línea Celular Tumoral , Supervivencia Celular , Medios de Cultivo Condicionados/farmacología , Humanos , Masculino , Óxido Nítrico/metabolismo , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Bi-directional transcription of Human Endogenous Retroviruses (hERVs) is a common feature of autoimmunity, neurodegeneration and cancer. Higher rates of cancer incidence, neurodegeneration and autoimmunity but a lower prevalence of autoimmune diseases characterize elderly people. Although the re-expression of hERVs is commonly observed in different cellular models of senescence as a result of the loss of their epigenetic transcriptional silencing, the hERVs modulation during aging is more complex, with a peak of activation in the sixties and a decline in the nineties. What is clearly accepted, instead, is the impact of the re-activation of dormant hERV on the maintenance of stemness and tissue self-renewing properties. An innate cellular immunity system, based on the RLR-MAVS circuit, controls the degradation of dsRNAs arising from the transcription of hERV elements, similarly to what happens for the accumulation of cytoplasmic DNA leading to the activation of cGAS/STING pathway. While agonists and inhibitors of the cGAS-STING pathway are considered promising immunomodulatory molecules, the effect of the RLR-MAVS pathway on innate immunity is still largely based on correlations and not on causality. Here we review the most recent evidence regarding the activation of MDA5-RIG1-MAVS pathway as a result of hERV de-repression during aging, immunosenescence, cancer and autoimmunity. We will also deal with the epigenetic mechanisms controlling hERV repression and with the strategies that can be adopted to modulate hERV expression in a therapeutic perspective. Finally, we will discuss if the RLR-MAVS signalling pathway actively modulates physiological and pathological conditions or if it is passively activated by them.
Asunto(s)
Retrovirus Endógenos , Neoplasias , Anciano , Envejecimiento , Retrovirus Endógenos/genética , Humanos , Inmunidad Innata , Neoplasias/genética , Transducción de Señal/fisiologíaRESUMEN
Cationic porphyrins bearing an alkyl side chain of 14 (2b) or 18 (2d) carbons dramatically inhibit proliferation of pancreatic cancer cells following treatment with light. We have compared two different ways of delivering porphyrin 2d: either in free form or engrafted into palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes (L-2d). Cell cytometry shows that while free 2d is taken up by pancreatic cancer cells by active (endocytosis) and passive (membrane fusion) transports, L-2d is internalized solely by endocytosis. Confocal microscopy showed that free 2d co-localizes with the cell membrane and lysosomes, whereas L-2d partly co-localizes with lysosomes and ER. It is found that free 2d inhibits the KRAS-Nrf2-GPX4 axis and strongly triggers lipid peroxidation, resulting in cell death by ferroptosis. By contrast, L-2d does not affect the KRAS-Nrf2-GPX4 axis and activates cell death mainly through apoptosis. Overall, our study demonstrates for the first time that cationic alkyl porphyrins, which have a IC50 ~ 23 nM, activate a dual mechanism of cell death, ferroptosis and apoptosis, where the predominant form depends on the delivery mode.
Asunto(s)
Neoplasias Pancreáticas , Porfirinas , Apoptosis , Cationes , Humanos , Liposomas/química , Factor 2 Relacionado con NF-E2 , Neoplasias Pancreáticas/tratamiento farmacológico , Porfirinas/farmacología , Proteínas Proto-Oncogénicas p21(ras) , Neoplasias PancreáticasRESUMEN
Recent studies have proven that the genetic landscape of pancreatic cancer is dominated by the KRAS oncogene. Its transcription is controlled by a G-rich motif (called 32R) located immediately upstream of the TSS. 32R may fold into a G-quadruplex (G4) in equilibrium between two G4 conformers: G9T (T M = 61.2 °C) and G25T (T M = 54.7 °C). We found that both G4s bind to hnRNPA1 and its proteolytic fragment UP1, promoting several contacts with the RRM protein domains. 1D NMR analysis of DNA imino protons shows that, upon binding to UP1, G25T is readily unfolded at both 5' and 3' tetrads, while G9T is only partially unfolded. The impact of hnRNPA1 on KRAS expression was determined by comparing Panc-1 cells with two Panc-1 knockout cell lines in which hnRNPA1 was deleted by the CRISPR/Cas9 technology. The results showed that the expression of KRAS is inhibited in the knockout cell lines, indicating that hnRNPA1 is essential for the transcription of KRAS. In addition, the knockout cell lines, compared to normal Panc-1 cells, show a dramatic decrease in cell growth and capacity of colony formation. Pull-down and Western blot experiments indicate that conformer G25T is a better platform than conformer G9T for the assembly of the transcription preinitiation complex with PARP1, Ku70, MAZ, and hnRNPA1. Together, our data prove that hnRNPA1, being a key transcription factor for the activation of KRAS, can be a new therapeutic target for the rational design of anticancer strategies.
RESUMEN
The promoter of the Kirsten ras (KRAS) proto-oncogene contains, upstream of the transcription start site, a quadruplex-forming motif called 32R with regulatory functions. As guanine under oxidative stress can be oxidized to 8-oxoguanine (8OG), we investigated the capacity of glycosylases 8-oxoguanine glycosylase (OGG1) and endonuclease VIII-like 1 (Neil1) to excise 8OG from 32R, either in duplex or G-quadruplex (G4) conformation. We found that OGG1 efficiently excised 8OG from oxidized 32R in duplex but not in G4 conformation. By contrast, glycosylase Neil1 showed more activity on the G4 than the duplex conformation. We also found that the excising activity of Neil1 on folded 32R depended on G4 topology. Our data suggest that Neil1, besides being involved in base excision repair pathway (BER), could play a role on KRAS transcription.
Asunto(s)
Daño del ADN , ADN Glicosilasas/metabolismo , Reparación del ADN , G-Cuádruplex , Transcripción Genética , Línea Celular Tumoral , ADN/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Estrés Oxidativo , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
In pancreatic Panc-1 cancer cells, an increase of oxidative stress enhances the level of 7,8-dihydro-8-oxoguanine (8OG) more in the KRAS promoter region containing G4 motifs than in non-G4 motif G-rich genomic regions. We found that H2O2 stimulates the recruitment to the KRAS promoter of poly [ADP-ribose] polymerase 1 (PARP-1), which efficiently binds to local G4 structures. Upon binding to G4 DNA, PARP-1 undergoes auto PARylation and thus becomes negatively charged. In our view this should favor the recruitment to the KRAS promoter of MAZ and hnRNP A1, as these two nuclear factors, because of their isoelectric points >7, are cationic in nature under physiological conditions. This is indeed supported by pulldown assays which showed that PARP-1, MAZ, and hnRNP A1 form a multiprotein complex with an oligonucleotide mimicking the KRAS G4 structure. Our data suggest that an increase of oxidative stress in Panc-1 cells activates a ROS-G4-PARP-1 axis that stimulates the transcription of KRAS. This mechanism is confirmed by the finding that when PARP-1 is silenced by siRNA or auto PARylation is inhibited by Veliparib, the expression of KRAS is downregulated. When Panc-1 cells are treated with H2O2 instead, a strong up-regulation of KRAS transcription is observed.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Neoplasias Pancreáticas/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Regulación hacia Arriba , Bencimidazoles/farmacología , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , G-Cuádruplex , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Guanina/análogos & derivados , Guanina/metabolismo , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Humanos , Estrés Oxidativo , Neoplasias Pancreáticas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/química , Factores de Transcripción/metabolismoRESUMEN
A continuous state of oxidative stress during inflammation contributes to the development of 25% of human cancers. Epithelial and inflammatory cells release reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can damage DNA. ROS/RNS have biological implications in both chemoresistance and tumor recurrence. As several clinically employed anticancer drugs can generate ROS/RNS, we have addressed herein how inducible nitric oxide synthase and nitric oxide (iNOS/â¢NO) affect the molecular pathways implicated in the tumor response to oxidative stress. To mimic the oxidative stress associated with chemotherapy, we used a photosensitizer (pheophorbide a) that can generate ROS/RNS in a controlled manner. We investigated how iNOS/â¢NO modulates the tumor response to oxidative stress by involving the NF-κB and Nrf2 molecular pathways. We found that low levels of iNOS induce the development of a more aggressive tumor population, leading to survival, recurrence and resistance. By contrast, high levels of iNOS/â¢NO sensitize tumor cells to oxidative treatment, causing cell growth arrest. Our analysis showed that NF-κB and Nrf2, which are activated in response to oxidative stress, communicate with each other through RKIP. For this critical role, RKIP could be an interesting target for anticancer drugs. Our study provides insight into the complex signaling response of cancer cells to oxidative treatments as well as new possibilities for the rational design of new therapeutic strategies.
Asunto(s)
Óxido Nítrico/fisiología , Estrés Oxidativo/fisiología , Neoplasias de la Próstata/metabolismo , Fármacos Sensibilizantes a Radiaciones/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , Clorofila/análogos & derivados , Clorofila/toxicidad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/efectos de la radiación , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo II/efectos de la radiación , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Neoplasias de la Próstata/patología , Especies Reactivas de Oxígeno/efectos de la radiaciónRESUMEN
KRAS is one of the most mutated oncogenes and still considered an undruggable target. An alternative strategy would consist in targeting its gene rather than the protein, specifically the formation of G-quadruplexes (G4) in its promoter. G4 are secondary structures implicated in biological processes, which can be formed among G-rich DNA (or RNA) sequences. Here we have studied the major conformations of the commonly known KRAS 32R, or simply 32R, a 32 residue sequence within the KRAS Nuclease Hypersensitive Element (NHE) region. We have determined the structure of the two major stable conformers that 32R can adopt and which display slow equilibrium (>ms) with each other. By using different biophysical methods, we found that the nucleotides G9, G25, G28 and G32 are particularly implicated in the exchange between these two conformations. We also showed that a triad at the 3' end further stabilizes one of the G4 conformations, while the second conformer remains more flexible and less stable.
Asunto(s)
ADN/genética , G-Cuádruplex , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Sitios de Unión/genética , Dicroismo Circular , Humanos , Terapia Molecular Dirigida , Neoplasias/genética , Neoplasias/terapia , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidoresRESUMEN
Designing small molecules able to break down G4 structures in mRNA (RG4s) offers an interesting approach to cancer therapy. Here, we have studied cationic porphyrins (CPs) bearing an alkyl chain up to 12 carbons, as they bind to RG4s while generating reactive oxygen species upon photoirradiation. Fluorescence-activated cell sorting (FACS) and confocal microscopy showed that the designed alkyl CPs strongly penetrate cell membranes, binding to KRAS and NRAS mRNAs under low-abundance cell conditions. In Panc-1 cells, alkyl CPs at nanomolar concentrations promote a dramatic downregulation of KRAS and NRAS expression, but only if photoactivated. Alkyl CPs also reduce the metabolic activity of pancreatic cancer cells and the growth of a Panc-1 xenograft in SCID mice. Propidium iodide/annexin assays and caspase 3, caspase 7, and PARP-1 analyses show that these compounds activate apoptosis. All these data demonstrate that the designed alkyl CPs are efficient photosensitizers for the photodynamic therapy of ras-driven cancers.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , G-Cuádruplex/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Fármacos Fotosensibilizantes/uso terapéutico , Porfirinas/uso terapéutico , Animales , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Secuencia de Bases , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Femenino , GTP Fosfohidrolasas/genética , Genes ras/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Ratones SCID , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/síntesis química , Porfirinas/síntesis química , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN/química , ARN/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In highly proliferating cancer cells oncogenic mutations reprogram the metabolism and increase the production of reactive oxygen species (ROS). Cancer cells prevent ROS accumulation by upregulating antioxidant systems. Here we show that an increase of oxidative stress (ROS and singlet oxygen), generated by photoactivated TMPyP4, results in the upregulation of KRAS and Nrf2, the major regulator of the redox homeostasis. In agreement with a previous observation, the ectopic expression of KRAS G12D or G12â¯V is found to stimulate Nrf2. This suggests that ROS, KRAS and Nrf2 establish a molecular axis controlling the redox homeostasis in cancer cells. We found that this axis also modulates the function of the NF-kB/Snail/RKIP circuitry, regulating the survival and apoptosis pathways. Our data show that low ROS levels, obtained when Nrf2 is activated by KRAS, results in the upregulation of prosurvival Snail and simultaneous downregulation of proapoptotic RKIP: an expression pattern favouring cell proliferation. By contrast, high ROS levels, obtained when Nrf2 is inhibited by a small molecule (luteolin), favour apoptosis by upregulating proapoptotic RKIP and downregulating prosurvival Snail. The results of this study are useful to design efficient photodynamic therapy (PDT) against cancer. We hypothesize that cancer cells can be sensitized to PDT when the photosensitizer is used in the presence of an inhibitor of Nrf2 (adjuvant). To test this hypothesis, we used luteolin (3',4',5,7-tetrahydroflavone) as Nrf2 inhibitor, since it reduces the expression of Nrf2 and increases intracellular ROS. By means of colony formation and viability assays we found that when Nrf2 is inhibited, PDT shows an increase of efficiency up to 45%.
Asunto(s)
Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Factor 2 Relacionado con NF-E2/genética , Neoplasias/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
Nowadays, active targeting of nanotherapeutics is a challenging issue. Here, we propose a rational design of a ternary nanoassembly (SAP) composed of nonionic amphiphilic ß-cyclodextrins (amphiphilic CD) incorporating pheophorbide (Pheo) as a phototherapeutic and an adamantanyl-folic acid conjugate (Ada-FA) to target tumor cells overexpressing α-folate receptor (FR-α(+)). Dynamic light scattering and ζ-potential pointed out the presence of nanoassemblies bearing a negative surface charge (ζ = -51 mV). Morphology of SAP was investigated by atomic force microscopy and microphotoluminescence, indicating the presence of highly emissive near-spherical assemblies of about 280 nm in size. Complementary spectroscopic techniques such as ROESY-NMR, UV/vis and steady-state fluorescence revealed that the folic acid protrudes out of amphiphilic CD rims, prone for recognition with FR-α. Pheo was strongly loaded in the nanoassembly mostly in monomeric form, thus generating singlet oxygen (1O2) and consequentely showing phototherapeutic action. SAP remained stable until 2 weeks in aqueous solutions. Stability studies in biologically relevant media pointed out the ability of SAP to interact with serum proteins by means of the oligoethylenglycole fringe, without destabilization. Release experiments demonstrated the sustained release of Pheo from SAP in environments mimiking physiological conditions (â¼20% within 1 week), plausibly suggesting low Pheo leaking and high integrity of the assembly within 24 h, time spent on average to reach the target sites. Cellular uptake of SAP was confirmed by confocal microscopy, pointing out that SAP was internalized into the tumoral cells expressing FR-α more efficiently than SP. SAP showed improved phototoxicity in human breast MCF-7 cancer cells FR-α(+) (IC50 = 270 nM) with respect to human prostate carcinoma PC3 cells (IC50 = 700 nM) that express a low level of that receptor (FR-α(-)). Finally, an improved phototoxicity in FR-α(+) MCF-7 cells (IC50 = 270 nM) was assessed after treatment with SAP vs SP (IC50 = 600 nM) which was designed without Ada-FA as a targeting unit.
Asunto(s)
Ciclodextrinas , Sistemas de Liberación de Medicamentos , Ácido Fólico , Neoplasias , Fotoquimioterapia , Ciclodextrinas/química , Ciclodextrinas/farmacología , Ácido Fólico/química , Ácido Fólico/farmacología , Humanos , Células MCF-7 , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Células PC-3RESUMEN
Datasets reporting microRNA expression profiles in normal and cancer cells show that miR-216b is aberrantly downregulated in pancreatic ductal adenocarcinoma (PDAC). We found that KRAS, whose mutant G12D allele drives the pathogenesis of PDAC, is a target of miR-216b. To suppress oncogenic KRAS in PDAC cells, we designed single-stranded (ss) miR-216b mimics with unlocked nucleic acid (UNA) modifications to enhance their nuclease resistance. We prepared variants of ss-miR-216b mimics with and without a 5' phosphate group. Both variants strongly suppressed oncogenic KRAS in PDAC cells and inhibited colony formation in pancreatic cancer cells. We observed that the designed ss-miR-216b mimics engaged AGO2 to promote the silencing of KRAS. We also tested a new delivery strategy based on the use of palmityl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with ss-miR-216b conjugated with two palmityl chains and a lipid-modified cell penetrating peptide (TAT). These versatile nanoparticles suppressed oncogenic KRAS in PDAC cells.
Asunto(s)
Proteínas Argonautas/genética , Carcinoma Ductal Pancreático/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Regiones no Traducidas 3'/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/terapia , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Lípidos/química , Lípidos/farmacología , Liposomas/química , Liposomas/farmacología , MicroARNs/química , MicroARNs/farmacología , Nanopartículas/administración & dosificación , Nanopartículas/química , Páncreas/metabolismo , Páncreas/patología , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidoresRESUMEN
KRAS is one of the most mutated genes in human cancer. It is controlled by a G4 motif located upstream of the transcription start site. In this paper, we demonstrate that 8-oxoguanine (8-oxoG), being more abundant in G4 than in non-G4 regions, is a new player in the regulation of this oncogene. We designed oligonucleotides mimicking the KRAS G4-motif and found that 8-oxoG impacts folding and stability of the G-quadruplex. Dimethylsulphate-footprinting showed that the G-run carrying 8-oxoG is excluded from the G-tetrads and replaced by a redundant G-run in the KRAS G4-motif. Chromatin immunoprecipitation revealed that the base-excision repair protein OGG1 is recruited to the KRAS promoter when the level of 8-oxoG in the G4 region is raised by H2O2. Polyacrylamide gel electrophoresis evidenced that OGG1 removes 8-oxoG from the G4-motif in duplex, but when folded it binds to the G-quadruplex in a non-productive way. We also found that 8-oxoG enhances the recruitment to the KRAS promoter of MAZ and hnRNP A1, two nuclear factors essential for transcription. All this suggests that 8-oxoG in the promoter G4 region could have an epigenetic potential for the control of gene expression.
Asunto(s)
G-Cuádruplex , Guanina/análogos & derivados , Proteínas Proto-Oncogénicas p21(ras)/química , Sitio de Iniciación de la Transcripción , Transcripción Genética , Línea Celular Tumoral , ADN/química , ADN/genética , ADN/metabolismo , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Guanina/química , Guanina/metabolismo , Células HEK293 , Ribonucleoproteína Nuclear Heterogénea A1/genética , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Humanos , Oxidación-Reducción , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The human KRAS transcript contains a G-rich 5'-UTR sequence (77% GC) harboring several G4 motifs capable to form stable RNA G-quadruplex (RG4) structures that can serve as targets for small molecules. A biotin-streptavidin pull-down assay showed that 4,11-bis(2-aminoethylamino)anthra[2,3-b]furan-5,10-dione (2a) binds to RG4s in the KRAS transcript under low-abundance cellular conditions. Dual-luciferase assays demonstrated that 2a and its analogue 4,11-bis(2-aminoethylamino)anthra[2,3-b]thiophene-5,10-dione (2b) repress translation in a dose-dependent manner. The effect of the G4-ligands on Panc-1 cancer cells has also been examined. Both 2a and 2b efficiently penetrate the cells, suppressing protein p21KRAS to <10% of the control. The KRAS down-regulation induces apoptosis together with a dramatic reduction of cell growth and colony formation. In summary, we report a strategy to suppress the KRAS oncogene in pancreatic cancer cells by means of small molecules binding to RG4s in the 5'-UTR of mRNA.
Asunto(s)
Regiones no Traducidas 5'/efectos de los fármacos , G-Cuádruplex/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Descubrimiento de Drogas , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Tiofenos/química , Tiofenos/farmacologíaRESUMEN
Photodynamic therapy (PDT) has drawn considerable attention for its efficacy against certain types of cancers. It shows however limits in the case of deep cancers, favoring tumor recurrence under suboptimal conditions. More insight into the molecular mechanisms of PDT-induced cytotoxicity and cytoprotection is essential to extend and strengthen this therapeutic modality. As PDT induces iNOS/NO in both tumor and microenvironment, we examined the role of nitric oxide (NO) in cytotoxicity and cytoprotection. Our findings show that NO mediates its cellular effects by acting on the NF-κB/YY1/RKIP loop, which controls cell growth and apoptosis. The cytoprotective effect of PDT-induced NO is observed at low NO levels, which activate the pro-survival/anti-apoptotic NF-κB and YY1, while inhibiting the anti-survival/pro-apoptotic and metastasis suppressor RKIP. In contrast, high PDT-induced NO levels inhibit NF-κB and YY1 and induce RKIP, resulting in significant anti-tumor activity. These findings reveal a critical role played by NO in PDT and suggest that the use of bifunctional PDT agents composed of a photosensitizer and a NO-donor could enhance the photo-treatment effect. A successful application of NO in anticancer therapy requires control of its concentration in the target tissue. To address this issue we propose as PDT agent, a bimolecular conjugate called DR2, composed of a photosensitizer (Pheophorbide a) and a non-steroidal anti-androgen molecule capable of releasing NO under the exclusive control of light. The mechanism of action of DR2 in prostate cancer cells is reported and discussed.
Asunto(s)
Clorofila/análogos & derivados , Donantes de Óxido Nítrico/farmacología , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Triazenos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Clorofila/farmacología , Humanos , Masculino , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Neoplasias de la Próstata/metabolismo , Factor de Transcripción YY1/metabolismoRESUMEN
KRAS is one of the most mutated genes in human cancer. Its crucial role in the tumourigenesis of pancreatic ductal adenocarcinoma (PDAC) has been widely demonstrated. As this deadly cancer does not sufficiently respond to conventional chemotherapies, it is important to increase our knowledge of pancreatic cancer biology, in particular how oncogenic KRAS is regulated. The promoter of KRAS contains a GA-element composed of runs of guanines that fold into a G4 structure. This unusual DNA conformation is recognized by several nuclear proteins, including MAZ and hnRNP A1. Recent data have revealed that KRAS is interconnected to ILK and hnRNP A1 in a circuitry that enables pancreatic cancer cells to maintain an aggressive phenotype. The present review illustrates recent advances on how KRAS is regulated in pancreatic cancer cells, focusing on the formation of G4 structures in the KRAS promoter and their interaction with hnRNP A1. The newly discovered KRAS-ILK-hnRNP A1 regulatory loop is discussed, emphasizing its potential as a therapeutic target for PDAC-specific molecules. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
Asunto(s)
ADN de Neoplasias/genética , G-Cuádruplex , Guanosina/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Transcripción Genética , Sitios de Unión , ADN de Neoplasias/química , ADN de Neoplasias/metabolismo , Regulación Neoplásica de la Expresión Génica , Guanosina/química , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Humanos , Ligandos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Polimorfismo Genético , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Relación Estructura-Actividad , Activación TranscripcionalRESUMEN
It is now well established that in the human genome the canonical double helix coexists with folded G-quadruplex structures that are known to have important biological functions. In this review we summarize the current knowledge on quadruplex formation in the promoters of the ras genes that are mutated in about 30% of all human cancers. We describe the nuclear proteins that recognize these unusual DNA structures and discuss their function in transcription. We also examine the formation of G-quadruplexes in the 5'-untranslated region of the ras transcripts and conclude this review by reporting strategies that use either ras G-quadruplexes or proteins recognizing the ras G-quadruplexes as targets of anticancer small molecules.
Asunto(s)
G-Cuádruplex , Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Transcripción Genética , Regiones no Traducidas 5'/genética , Genoma Humano , Humanos , Neoplasias/terapia , Regiones Promotoras Genéticas , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
Cationic porphyrins (Prs) and phthalocyanines (Pcs) are strong photosensitizers that have drawn much attention for their potential in photodynamic therapy. These compounds have the interesting property of binding to nucleic acids, in particular G-rich quadruplex-forming sequences in DNA and RNA. In this review, we highlight their potential as anticancer drugs.