RESUMEN
Two novel cyclometallated iridium(III) complexes have been prepared with one bidentate or two monodentate imidazole-based ligands, 1 and 2, respectively. The complexes showed intense emission with long lifetimes of the excited state. Femtosecond transient absorption experiments established the nature of the lowest excited state as 3IL state. Singlet oxygen generation with good yields (40% for 1 and 82% for 2) was established by detecting 1O2 directly, through its emission at 1270 nm. Photostability studies were also performed to assess the viability of the complexes as photosensitizers (PS) for photodynamic therapy (PDT). Complex 1 was selected as a good candidate to investigate light-activated killing of cells, whilst complex 2 was found to be toxic in the dark and unstable under light. Complex 1 demonstrated high phototoxicity indexes (PI) in the visible region, PI > 250 after irradiation at 405 nm and PI > 150 at 455 nm, in EJ bladder cancer cells.
Asunto(s)
Bencimidazoles , Neoplasias , Fotoquimioterapia , Ligandos , Línea Celular Tumoral , Fármacos Fotosensibilizantes/química , Muerte Celular , Iridio/farmacología , Iridio/químicaRESUMEN
Macromolecules are potentially useful delivery systems for cancer drugs, as their size allows them to utilize the enhanced permeability and retention effect (EPR), which facilitates selective delivery to (and retention within) tumors. In addition, macromolecular delivery systems can prolong circulation times as well as protect and solubilize toxic and hydrophobic drug moieties. Overall, these properties and abilities can result in an enhanced therapeutic effect. Photodynamic therapy (PDT) combines the use of oxygen and a photosensitizer (PS), which become toxic upon light irradiation. We proposed that a PS encapsulated within a water-soluble macromolecule could exploit the EPR effect and safely and selectively deliver the PS to a tumor. In this paper, we describe the synthesis of a porphyrin-cored hyperbranched polymer that aggregated into larger micellar structures. DLS and TEM indicated that these aggregated structures had diameters of 45 and 20 nm for the solvated and nonsolvated species, respectively. The porphyrin-cored HBP (PC-HBP), along with the nonencapsulated porphyrin (THPP), were screened against EJ bladder carcinoma cells in the dark and light. Both THPP and PC-HBP displayed good toxicity in the light, with LD50 concentrations of 0.5 and 1.7 µM, respectively. However, in the dark, the nonincorporated porphyrin (THPP) displayed significant toxicity, generating an LD50 of 4 µM. On the other hand, no dark toxicity was observed for the polymer system (PC-HBP) at concentrations of 100 µM or less. As such, incorporation within the large polymer aggregate serves to eliminate dark toxicity while maintaining excellent toxicity when irradiated.
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Micelas , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Porfirinas/química , Glicoles de Propileno/química , Neoplasias de la Vejiga Urinaria/terapia , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Oscuridad , Composición de Medicamentos/métodos , Sistemas de Liberación de Medicamentos , Humanos , Microscopía Confocal , Nanosferas , Tamaño de la Partícula , Polímeros/uso terapéutico , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
The series of complexes [Os(bpy)3- n(pytz) n][PF6]2 (bpy = 2,2'-bipyridyl, pytz = 1-benzyl-4-(pyrid-2-yl)-1,2,3-triazole, 1 n = 0, 2 n = 1, 3 n = 2, 4 n = 3) were prepared and characterized and are rare examples of luminescent 1,2,3-triazole-based osmium(II) complexes. For 3 we present an attractive and particularly mild preparative route via an osmium(II) η6-arene precursor circumventing the harsh conditions that are usually required. Because of the high spin-orbit coupling constant associated with the Os(II) center the absorption spectra of the complexes all display absorption bands of appreciable intensity in the range of 500-700 nm corresponding to spin-forbidden ground-state-to-3MLCT transitions (MLCT = metal-to-ligand charge transfer), which occur at significantly lower energies than the corresponding spin-allowed 1MLCT transitions. The homoleptic complex 4 is a bright emitter (λmaxem = 614 nm) with a relatively high quantum yield of emission of â¼40% in deoxygenated acetonitrile solutions at room temperature. Water-soluble chloride salts of 1-4 were also prepared, all of which remain emissive in aerated aqueous solutions at room temperature. The complexes were investigated for their potential as phosphorescent cellular imaging agents, whereby efficient excitation into the 3MLCT absorption bands at the red side of the visible range circumvents autofluorescence from biological specimens, which do not absorb in this region of the spectrum. Confocal microscopy reveals 4 to be readily taken up by cancer cell lines (HeLa and EJ) with apparent lysosomal and endosomal localization, while toxicity assays reveal that the compounds have low dark and light toxicity. These complexes therefore provide an excellent platform for the development of efficient luminescent cellular imaging agents with advantageous photophysical properties that enable excitation and emission in the biologically transparent region of the optical spectrum.
Asunto(s)
Complejos de Coordinación/química , Sustancias Luminiscentes/química , Imagen Óptica , Osmio/química , Piridinas/química , Triazoles/química , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/farmacología , Células HeLa , Humanos , Sustancias Luminiscentes/síntesis química , Sustancias Luminiscentes/farmacología , Mediciones Luminiscentes , Estructura Molecular , Procesos Fotoquímicos , Teoría Cuántica , Células Tumorales CultivadasRESUMEN
Photodynamic therapy (PDT) uses photosensitizers (PS) which only become cytotoxic upon light-irradiation. Transition-metal complexes are highly promising PS due to long excited-state lifetimes, and high photo-stabilities. However, these complexes usually absorb higher-energy UV/Vis light, whereas the optimal tissue transparency is in the lower-energy NIR region. Two-photon excitation (TPE) can overcome this dichotomy, with simultaneous absorption of two lower-energy NIR-photons populating the same PS-active excited state as one higher-energy photon. We introduce two low-molecular weight, long-lived and photo-stable iridium complexes of the [Ir(N^C)2 (N^N)]+ family with high TP-absorption, which localise to mitochondria and lysosomal structures in live cells. The compounds are efficient PS under 1-photon irradiation (405â nm) resulting in apoptotic cell death in diverse cancer cell lines at low light doses (3.6â J cm-2 ), low concentrations, and photo-indexes greater than 555. Remarkably 1 also displays high PS activity killing cancer cells under NIR two-photon excitation (760â nm), which along with its photo-stability indicates potential future clinical application.
Asunto(s)
Complejos de Coordinación/farmacología , Iridio/farmacología , Neoplasias/tratamiento farmacológico , Fármacos Fotosensibilizantes/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Complejos de Coordinación/química , Humanos , Iridio/química , Luz , Neoplasias/metabolismo , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Oxígeno Singlete/metabolismoRESUMEN
RECQL5 is a member of the RecQ family of DNA helicases and has key roles in homologous recombination, base excision repair, replication and transcription. The clinicopathological significance of RECQL5 expression in breast cancer is unknown. In this study, we have evaluated RECQL5 mRNA expression in 1977 breast cancers, and RECQL5 protein level in 1902 breast cancers [Nottingham Tenovus series (n = 1650) and ER- cohort (n = 252)]. Expression levels were correlated to aggressive phenotypes and survival outcomes. High RECQL5 mRNA expression was significantly associated with high histological grade (P = 0.007), HER2 overexpression (P = 0.032), ER+/HER2-/high proliferation genefu subtype (P < 0.0001), integrative molecular clusters (intClust 1and 9) (P < 0.0001) and poor survival (P < 0.0001). In subgroup analysis, high RECQL5 mRNA level remains significantly associated with poor BCSS in ER+ cohort (P < 0.0001) but not in ER- cohort (P = 0.116). At the protein level, in tumours with low RAD51, high RECQL5 level was significantly associated with high histological grade (P < 0.0001), higher mitotic index (P = 0.008), dedifferentiation (P = 0.025), pleomorphism (P = 0.027) and poor survival (P = 0.003). In subgroup analysis, high RECQL5/low RAD51 remains significantly associated with poor BCSS in ER+ cohort (P = 0.010), but not in ER- cohort (P = 0.628). In multivariate analysis, high RECQL5 mRNA and high RECQL5/low RAD51 nuclear protein coexpression independently influenced survival (P = 0.022) in whole cohort and in the ER+ subgroup. Preclinically, we show that exogenous expression of RECQL5 in MCF10A cells can drive proliferation supporting an oncogenic function for RECQL5 in breast cancer. We conclude that RECQL5 is a promising biomarker in breast cancer.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , RecQ Helicasas/biosíntesis , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , ADN-Topoisomerasas de Tipo II/biosíntesis , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Células MCF-7 , Invasividad Neoplásica , Pronóstico , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Recombinasa Rad51/biosíntesis , Recombinasa Rad51/genética , RecQ Helicasas/genética , Análisis de Matrices TisularesRESUMEN
Luminescent, mixed metal d-f complexes have the potential to be used for dual (magnetic resonance imaging (MRI) and luminescence) in vivo imaging. Here, we present dinuclear and trinuclear d-f complexes, comprising a rigid framework linking a luminescent Ir center to one (Ir·Ln) or two (Ir·Ln2) lanthanide metal centers (where Ln = Eu(III) and Gd(III), respectively). A range of physical, spectroscopic, and imaging-based properties including relaxivity arising from the Gd(III) units and the occurrence of Ir(III) â Eu(III) photoinduced energy-transfer are presented. The rigidity imposed by the ligand facilitates high relaxivities for the Gd(III) complexes, while the luminescence from the Ir(III) and Eu(III) centers provide luminescence imaging capabilities. Dinuclear (Ir·Ln) complexes performed best in cellular studies, exhibiting good solubility in aqueous solutions, low toxicity after 4 and 18 h, respectively, and punctate lysosomal staining. We also demonstrate the first example of oxygen sensing in fixed cells using the dyad Ir·Gd, via two-photon phosphorescence lifetime imaging (PLIM).
Asunto(s)
Iridio/química , Lantano/química , Sondas Moleculares , Oxígeno/análisis , Línea Celular Tumoral , Humanos , Luminiscencia , Espectrofotometría UltravioletaRESUMEN
The complex [Os(btzpy)2][PF6]2 (1, btzpy = 2,6-bis(1-phenyl-1,2,3-triazol-4-yl)pyridine) has been prepared and characterised. Complex 1 exhibits phosphorescence (λem = 595 nm, τ = 937 ns, φem = 9.3% in degassed acetonitrile) in contrast to its known ruthenium(II) analogue, which is non-emissive at room temperature. The complex undergoes significant oxygen-dependent quenching of emission with a 43-fold reduction in luminescence intensity between degassed and aerated acetonitrile solutions, indicating its potential to act as a singlet oxygen sensitiser. Complex 1 underwent counterion metathesis to yield [Os(btzpy)2]Cl2 (1Cl), which shows near identical optical absorption and emission spectra to those of 1. Direct measurement of the yield of singlet oxygen sensitised by 1Cl was carried out (φ (¹O2) = 57%) for air equilibrated acetonitrile solutions. On the basis of these photophysical properties, preliminary cellular uptake and luminescence microscopy imaging studies were conducted. Complex 1Cl readily entered the cancer cell lines HeLa and U2OS with mitochondrial staining seen and intense emission allowing for imaging at concentrations as low as 1 µM. Long-term toxicity results indicate low toxicity in HeLa cells with LD50 >100 µM. Osmium(II) complexes based on 1 therefore present an excellent platform for the development of novel theranostic agents for anticancer activity.
Asunto(s)
Mitocondrias/ultraestructura , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/farmacología , Osmio/química , Triazoles/síntesis química , Triazoles/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células HeLa , Humanos , Mediciones Luminiscentes , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Moleculares , Estructura Molecular , Compuestos Organometálicos/química , Oxígeno Singlete/química , Solubilidad , Nanomedicina Teranóstica , Triazoles/química , Agua/químicaRESUMEN
Neuroblastoma is the most common extracranial cancer in childhood. High-risk neuroblastoma continues to have a poor prognosis and there is an urgent need to design biologically based therapies that specifically target the pathways responsible for malignant transformation and progression. One such pathway is the PI3K/Akt/mTOR pathway. In this article we outline the evidence for aberrant activation of the PI3K/Akt/mTOR pathway in neuroblastoma and discuss the possible mechanisms which mediate it. We also discuss the development of treatments targeting this pathway in neuroblastoma and the challenges that must be overcome before such treatments can enter routine clinical practice.
Asunto(s)
Neuroblastoma/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/antagonistas & inhibidores , Neuroblastoma/etiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/fisiologíaRESUMEN
Complex DNA damage (CDD), containing two or more DNA lesions within one or two DNA helical turns, is a signature of ionising radiation (IR) and contributes significantly to the therapeutic effect through cell killing. The levels and complexity of CDD increases with linear energy transfer (LET), however, the specific cellular response to this type of DNA damage and the critical proteins essential for repair of CDD is currently unclear. We performed an siRNA screen of ~240 DNA damage response proteins to identify those specifically involved in controlling cell survival in response to high-LET protons at the Bragg peak, compared to low-LET entrance dose protons which differ in the amount of CDD produced. From this, we subsequently validated that depletion of 8-oxoguanine DNA glycosylase (OGG1) and poly(ADP-ribose) glycohydrolase (PARG) in HeLa and head and neck cancer cells leads to significantly increased cellular radiosensitivity specifically following high-LET protons, whilst no effect was observed after low-LET protons and X-rays. We subsequently confirmed that OGG1 and PARG are both required for efficient CDD repair post-irradiation with high-LET protons. Importantly, these results were also recapitulated using specific inhibitors for OGG1 (TH5487) and PARG (PDD00017273). Our results suggest OGG1 and PARG play a fundamental role in the cellular response to CDD and indicate that targeting these enzymes could represent a promising therapeutic strategy for the treatment of head and neck cancers following high-LET radiation.
Asunto(s)
ADN Glicosilasas , Neoplasias de Cabeza y Cuello , Humanos , Protones , Transferencia Lineal de Energía , Daño del ADN , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/radioterapia , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismoRESUMEN
Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.
Asunto(s)
Diferenciación Celular , Variaciones en el Número de Copia de ADN , Proteína Proto-Oncogénica N-Myc , Cresta Neural , Neuroblastoma , Humanos , Neuroblastoma/genética , Neuroblastoma/patología , Cresta Neural/metabolismo , Cresta Neural/patología , Femenino , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Aberraciones Cromosómicas , Células Madre Embrionarias Humanas/metabolismo , Transcriptoma , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
INTRODUCTION: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.
Asunto(s)
Neoplasias de la Mama , Investigación , Investigación Biomédica Traslacional , Animales , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/etiología , Neoplasias de la Mama/terapia , Femenino , HumanosRESUMEN
If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea-induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Recombinación Genética/fisiología , Animales , Células Cultivadas , Cricetinae , Cricetulus , Reparación del ADN , Técnica del Anticuerpo FluorescenteRESUMEN
Tumour survival and growth are reliant on angiogenesis, the formation of new blood vessels, to facilitate nutrient and waste exchange and, importantly, provide a route for metastasis from a primary to a secondary site. Whilst current models can ensure the transport and exchange of nutrients and waste via diffusion over distances greater than 200 µm, many lack sufficient vasculature capable of recapitulating the tumour microenvironment and, thus, metastasis. In this study, we utilise gelatin-containing polymerised high internal phase emulsion (polyHIPE) templated polycaprolactone-methacrylate (PCL-M) scaffolds to fabricate a composite material to support the 3D culture of MDA-MB-231 breast cancer cells and vascular ingrowth. Firstly, we investigated the effect of gelatin within the scaffolds on the mechanical and chemical properties using compression testing and FTIR spectroscopy, respectively. Initial in vitro assessment of cell metabolic activity and vascular endothelial growth factor expression demonstrated that gelatin-containing PCL-M polyHIPEs are capable of supporting 3D breast cancer cell growth. We then utilised the chick chorioallantoic membrane (CAM) assay to assess the angiogenic potential of cell-seeded gelatin-containing PCL-M polyHIPEs, and vascular ingrowth within cell-seeded, surfactant and gelatin-containing scaffolds was investigated via histological staining. Overall, our study proposes a promising composite material to fabricate a substrate to support the 3D culture of cancer cells and vascular ingrowth.
RESUMEN
Chemoresistance poses a great barrier to breast cancer treatment and is thought to correlate with increased matrix stiffness. We developed two-dimensional (2D) polyacrylamide (PAA) and three-dimensional (3D) alginate in vitro models of tissue stiffness that mimic the stiffness of normal breast and breast cancer. We then used these to compare cell viability in response to chemotherapeutic treatment. In both 2D and 3D we observed that breast cancer cell growth and size was increased at a higher stiffness corresponding to tumours compared to normal tissue. When chemotherapeutic response was measured, a specific differential response in cell viability was observed for gemcitabine in 2 of the 7 breast cancer cell lines investigated. MCF7 and T-47D cell lines showed gemcitabine resistance at 4 kPa compared to 500 Pa. These cell lines share a common phenotype of progesterone receptor (PGR) expression and, indeed, pre-treatment with the selective progesterone receptor modulator (SPRM) mifepristone abolished resistance to gemcitabine at high stiffness. Our data reveals that combined treatment with SPRMs may therefore help in reducing resistance to gemcitabine in stiffer breast tumours which are PGR positive.
Asunto(s)
Neoplasias de la Mama , Receptores de Progesterona , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Femenino , Humanos , Progesterona/uso terapéutico , Receptores de Progesterona/metabolismo , GemcitabinaRESUMEN
The PARP-3 protein is closely related to the PARP-1 and PARP-2 proteins, which are involved in DNA repair and genome maintenance. Here, we characterized the biochemical properties of human PARP-3. PARP-3 is able to ADP-ribosylate itself as well as histone H1, a previously unknown substrate for PARP-3. PARP-3 is not activated upon binding to DNA and is a mono-ADP-ribosylase, in contrast to PARP-1 and PARP-2. PARP-3 interacts with PARP-1 and activates PARP-1 in the absence of DNA, resulting in synthesis of polymers of ADP-ribose. The N-terminal WGR domain of PARP-3 is involved in this activation. The functional interaction between PARP-3 and PARP-1 suggests that it may have a role in DNA repair. However, here we report that PARP-3 small interfering RNA-depleted cells are not sensitive to the topoisomerase I poison camptothecin, inducing DNA single-strand breaks, and repair these lesions as efficiently as wild-type cells. Altogether, these results suggest that the interaction between PARP-1 and PARP-3 is unrelated to DNA single-strand break repair.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Cadena Simple , Reparación del ADN/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , 3-Yodobencilguanidina/farmacología , Camptotecina/farmacología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Inhibidores Enzimáticos/farmacología , Histonas/metabolismo , Humanos , Hidrólisis , Hidroxilamina/farmacología , Cloruro de Mercurio/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/genética , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Inhibidores de Topoisomerasa IRESUMEN
Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage. Nevertheless, PARP1-/- mice are viable, fertile and do not develop early onset tumours. Here, we show that PARP inhibitors trigger gamma-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2-/-) in BRCA2+/- patients are defective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment.
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Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proteína BRCA2/deficiencia , Genes BRCA2 , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Azulenos , Proteína BRCA2/genética , Benzodiazepinas/farmacología , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Replicación del ADN , Ratones , Ratones Desnudos , Neoplasias/enzimología , Neoplasias/patología , Poli(ADP-Ribosa) Polimerasas/deficiencia , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Arsenic exposure constitutes one of the most widespread environmental carcinogens, and is associated with increased risk of many different types of cancers. Here we report that arsenite (As[III]) can induce both replication-dependent DNA double-strand breaks (DSB) and homologous recombination (HR) at doses as low as 5 microM (0.65 mg/l), which are within the typical doses often found in drinking water in contaminated areas. We show that the production of DSBs is dependent on active replication and is likely to be the result of conversion of a DNA single-strand break (SSB) into a toxic DSB when encountered by a replication fork. We demonstrate that HR is required for the repair of these breaks and show that a functional HR pathway protects against As[III]-induced cytotoxicity. In addition, BRCA2-deficient cells are sensitive to As[III] and we suggest that As[III] could be exploited as a therapy for HR-deficient tumours such as BRCA1 and BRCA2 mutated breast and ovarian cancers.
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Arsenitos/toxicidad , Proteína BRCA2/metabolismo , Carcinógenos/toxicidad , Roturas del ADN de Doble Cadena , Reparación del ADN , Recombinación Genética , Animales , Línea Celular , Cricetinae , Cricetulus , Replicación del ADNRESUMEN
Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.
Asunto(s)
Reactivos de Enlaces Cruzados/toxicidad , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Recombinación Genética , Animales , Línea Celular , Cisplatino/toxicidad , Cricetinae , Roturas del ADN de Doble Cadena , Replicación del ADN/efectos de los fármacos , Humanos , Mitomicina/toxicidad , Mutación , Recombinasa Rad51/genéticaRESUMEN
The evaluation of novel photosensitizers (PSs) for photodynamic therapy (PDT) is difficult due to the limitations of two-dimensional cell culture and multiple parameters (dose, light intensity, uptake time), which complicate progression to in vivo experiments and clinical translation. Three-dimensional (3D) cell culture models like multicellular cancer tumor spheroids (MCTS) show great similarities to in vivo avascular tumor conditions, improving the speed and accuracy of screening novel compounds with various treatment combinations. In this study, we utilize C8161 human melanoma spheroids to screen PDT treatment combinations using protoporphyrin IX (PpIX) and drug-loaded carbon dot (CD) conjugates PpIX-CD and PpIX@CD at ultralow fluence values (<10 J/cm2). Conjugates show equivalent light-induced damage to PpIX from 1 µg/mL with significantly less dark cytotoxicity up to 72 h after exposure, shown by LDH release and dsDNA content. Fractionated treatments, carried out by dividing light exposure with 24 h intervals, demonstrate an enhanced PDT effect compared to single exposure at equal concentrations. Light sheet fluorescence microscopy combined with live/dead staining demonstrates that spheroids sustain extensive damage after PDT, with PpIX and PpIX-CD showing improved uptake compared to PpIX@CD. We show that PDT parameter screening can be carried out using a low-cost and convenient combination of assays to improve the efficiency of evaluating novel compounds.
Asunto(s)
Dermatitis Fototóxica , Melanoma , Fotoquimioterapia , Ácido Aminolevulínico , Humanos , Melanoma/tratamiento farmacológico , Fármacos Fotosensibilizantes/farmacologíaRESUMEN
Photodynamic therapy (PDT) is a treatment which uses light-activated compounds to produce reactive oxygen species, leading to membrane damage and cell death. Multicellular cancer spheroids are a preferable alternative for PDT evaluation in comparison to monolayer cell cultures due to their ability to better mimic in vivo avascular tumour characteristics such as hypoxia and cell-cell interactions, low cost, and ease of production. However, inconsistent growth kinetics and drug responsiveness causes poor experimental reproducibility and limits their usefulness. Herein, we used image analysis to establish a link between human melanoma C8161 spheroid morphology and drug responsiveness. Spheroids were pre-selected based on sphericity, area, and diameter, reducing variation in experimental groups before treatment. Spheroid morphology after PDT was analyzed using AnaSP and ReViSP, MATLAB-based open-source software, obtaining nine different parameters. Spheroids displayed a linear response between biological assays and morphology, with area (R2 = 0.7219) and volume (R2 = 0.6138) showing the best fit. Sphericity, convexity, and solidity were confirmed as poor standalone indicators of spheroid viability. Our results indicate spheroid morphometric parameters can be used to accurately screen inefficient treatment combinations of novel compounds.