RESUMEN
Ferrocifens are an original class of ferrocifen-type breast cancer drugs. They possess anti-proliferative effects due to the association of the ferrocene moiety and the tamoxifen skeleton. In this work, fluorescence measurements indicated the production of reactive oxygen species (ROS) if hormone-dependent or -independent breast cancer cells were incubated with three hit ferrocifen compounds. Additionally, amperometry at ultramicroelectrodes was carried out to identify and quantify ROS and reactive nitrogen species (RNS) under stress conditions. Videomicroscopy was used to optimize the conditions employed for electrochemical investigations. Amperometry was then performed on two cell lines pre-incubated with each of the three ferrocifens. Interestingly, these results demonstrate that the presence of an aminoalkyl chain in the ferrocifen structure may confer a unique behavior toward both cell lines, in comparison with the two other compounds that lack this feature.
Asunto(s)
Compuestos Ferrosos/química , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Técnicas Electroquímicas , Electrodos , Femenino , Compuestos Ferrosos/toxicidad , Humanos , Células MCF-7 , Microscopía por VideoRESUMEN
PURPOSE: Epidermal growth factor receptor (EGFR) is critically involved in progression and therapy resistance of squamous cell carcinoma (SCC). Albeit EGFR targeting could improve the effect of radiotherapy on patients' outcome, the clinical results failed to meet expectations from preclinical studies. In this work, we evaluated the potential of the radionuclide Yttrium-90 ((90)Y) bound to Cetuximab ((90)Y-Cetuximab) as novel targeting approach for SCC cells in vitro. MATERIALS AND METHODS: FaDu and A431 cell lines were used. EGFR subcellular localization, clonogenic survival, radiation-induced γH2AX foci and EGFR signaling were examined. Cells were treated with DTPA, DTPA-Cetuximab, (90)Y and (90)Y-Cetuximab alone or in combination with external X-ray irradiation. RESULTS: Dose- and cell line-dependently, (90)Y-Cetuximab mediated a significant reduction in clonogenicity relative to unbound (90)Y. Combined 2-Gy external radiation plus 2-Gy equivalent dose of (90)Y-Cetuximab was more effective than equivalent doses of (90)Y and X-ray radiation. Analogous effects were observed in the number of residual radiation-induced foci. Additionally, EGFR, ERK1/2 and AKT phosphorylation showed alterations upon different treatments. CONCLUSIONS: Our findings show that Cetuximab-conjugated (90)Y has a significant potential to eradicate human SCC cells. A combination of radioimmunotherapeutic compounds and external radiotherapy might be a promising treatment strategy for clinical application.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de Cabeza y Cuello/patología , Recuento de Células , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cetuximab , Terapia Combinada , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Espacio Intracelular/efectos de la radiación , Terapia Molecular Dirigida , Ácido Pentético/química , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/efectos de la radiación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Radioisótopos de Itrio/química , Radioisótopos de Itrio/uso terapéuticoRESUMEN
We have designed two novel dendrimers with cyclam cores with appended poly(amido amine) (PAMAM) dendrons, decorated at the periphery with four and eight dansyl chromophores, respectively. The photophysical properties of the dendrimers and their Nd(3+) complexes have been investigated. The energy-transfer efficiency to the lanthanide ions from these dendrimers has been studied as a function of the generation. It has been observed that an increase in the dendrimer generation as well as the number of amide units enhances the energy transfer to the lanthanide ion.
Asunto(s)
Dendrímeros/química , Elementos de la Serie de los Lantanoides/química , Neodimio/química , Complejos de Coordinación/química , Compuestos de Dansilo/química , Dendrímeros/síntesis química , Iones/química , Ligandos , Espectrometría de FluorescenciaRESUMEN
UNLABELLED: Radioimmunotherapy is considered to have great potential for efficient and highly specific treatment of tumors. The aim of this study was to determine the efficacy of radioimmunotherapy when using (90)Y-labeled cetuximab and to determine to what degree induction and repair of DNA double-strand breaks (DSBs) are decisive for this approach. METHODS: This study was performed with 9 cell lines of squamous cell carcinoma of the head and neck (HNSCC) differing strongly in epidermal growth factor receptor (EGFR) expression. The radionuclide (90)Y was coupled by the chelator trans-cyclohexyl-diethylene-triamine-pentaacetic acid (CHX-Aâ³-DTPA)/linker construct to the EGFR-directed antibody cetuximab to yield (90)Y-Y-CHX-Aâ³-DTPA-cetuximab with a specific activity of approximately 1.2 GBq/mg. EGFR expression was determined by immunofluorescence and Western blotting, cetuximab binding by fluorescence-activated cell sorter analysis, the number of DSBs by immunofluorescence staining γH2AX/53BP1-positive repair foci, and cell survival by colony formation. RESULTS: For the 9 HNSCC cell lines, cetuximab binding correlated with the amount of EGFR present in the cell membrane (r(2) = 0.967, P < 0.001). When cells were exposed to (90)Y-Y-CHX-Aâ³-DTPA-cetuximab, the number of induced DSBs increased linearly with time (r(2) = 0.968, P = 0.016). This number was found to correlate with the amount of membranous EGFR (r(2) = 0.877, P = 0.006). Most DSBs were repaired during incubation at 37°C, but the small number of remaining DSBs still correlated with the amount of membranous EGFR (24 h: r(2) = 0.977, P < 0.001; 48 h: r(2) = 0.947, P < 0.001). Exposure to (90)Y-Y-CHX-Aâ³-DTPA-cetuximab also resulted in efficient cell killing, whereby the extent of cell killing correlated strongly with the respective number of remaining DSBs (r(2) = 0.989, P < 0.001) and with the amount of membranous EGFR (r(2) = 0.967, P < 0.001). No cell killing was observed for UTSCC15 cells with low EGFR expression, in contrast to the strong reduction of 86% measured for UTSCC14 cells showing a strong overexpression of EGFR. CONCLUSION: (90)Y-Y-CHX-Aâ³-DTPA-cetuximab affected cell survival through the induction of DSBs. This treatment was especially efficient for HNSCC cells strongly overexpressing EGFR, whereas no effect was seen for cells with low levels of EGFR expression. Therefore, EGFR-directed radioimmunotherapy using (90)Y-Y-CHX-Aâ³-DTPA-cetuximab appears to be a powerful tool that can be used to inactivate tumors with strong EGFR overexpression, which are often characterized by a pronounced radioresistance.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Carcinoma de Células Escamosas/radioterapia , Neoplasias de Cabeza y Cuello/radioterapia , Inmunoconjugados/uso terapéutico , Radioisótopos de Itrio/uso terapéutico , Anticuerpos Monoclonales Humanizados , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Muerte Celular/efectos de la radiación , Línea Celular Tumoral , Cetuximab , Roturas del ADN de Doble Cadena , Reparación del ADN , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , ARN Interferente Pequeño/genética , Tolerancia a Radiación , RadioinmunoterapiaRESUMEN
The aim of this work was to investigate the mechanism of action of ferrocifen (Fc-OH-TAM), the ferrocenyl analog of 4-hydroxy-tamoxifen (OH-TAM), which is the active metabolite of tamoxifen, the drug most widely prescribed for treatment of hormone-dependent breast cancers. Fc-OH-TAM showed an anti-proliferative effect on the six breast cancer cell lines tested, 3 ERalpha positive (MCF-7, T-47D, ZR-75-1) and 3 ERalpha negative (MDA-MB-231, SKBR-3, Hs578-T) whatever their ER (estrogen receptor) status. However, the mechanism of action of the ferrocenyl derivative appeared to differ depending on the status of the ERalpha. Analysis of cell cycle distribution revealed that Fc-OH-TAM first recruits cells in the S phase in both ERalpha positive and ERalpha negative cells. In the presence of ERalpha, Fc-OH-TAM allowed cell cycle progression, with a subsequent blockade in G0/G1, whereas in the absence of ERalpha, cells remained in the S phase. Significant production of ROS was observed only in the presence of Fc-OH-TAM in both ERalpha positive and negative breast cancer cell lines. Within our experimental conditions, this ROS production is associated with cell cycle arrest and senescence rather than apoptosis. In the presence of ERalpha, Fc-OH-TAM seems to mainly act in the same way as OH-TAM but also induces an additional cytotoxic effect not mediated by the receptor. Our data suggest that this cytotoxic effect of Fc-OH-TAM is expressed via a mechanism of action distinct from the non-genomic pathway observed with high doses of OH-Tamoxifen.
Asunto(s)
Neoplasias de la Mama/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Compuestos Ferrosos/metabolismo , Compuestos Ferrosos/farmacología , Receptores de Estrógenos/metabolismo , Tamoxifeno/análogos & derivados , Acetilcisteína/metabolismo , Antioxidantes/metabolismo , Ciclo Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Femenino , Compuestos Ferrosos/química , Humanos , Especies Reactivas de Oxígeno/metabolismo , Receptores de Estrógenos/genética , Tamoxifeno/química , Tamoxifeno/metabolismo , Tamoxifeno/farmacologíaRESUMEN
Hydroxyferrocifen compounds are a new and promising class of ferrocifen-type breast-cancer drug candidates. They possess both endocrine-modulating properties and cytotoxic activity, which come from the tamoxifen skeleton and the presence of a ferrocene moiety, respectively. However, they suffer from reduced solubility in water, which presents a problem for their eventual therapeutic use. Herein, we examined the interactions of hydroxyferrocifen compounds with cyclodextrins (CDs) to evaluate whether or not their electron-transfer oxidation pathways were affected by their inclusion. It has been demonstrated that these inclusion complexes are soluble in pure water, which shows that CDs can be used to deliver these biologically active molecules. Therefore, a series of these compounds has been investigated by cyclic voltametry in various media in the presence of CDs (beta-CD and Me-beta-CD). In methanol, the hydroxyferrocifen compounds exhibited a weak interaction with the CD cavity. These interactions became stronger as the amount of added water increased. The complexation effect between the hydroxyferrocifen compounds and beta-CD was found to be stronger if the CD was partially methylated, which is probably due to hydrophobic effects between the cyclopentadienyl ring and/or the aromatic rings and the methoxy groups. Moreover, it appears that the structure of the hydroxyferrocifen compounds affects both their solubility and their complexation dynamics. Investigations in the presence of pyridine show that the base kinetically favors the dissociation of the ferrocifen-CD complex during the electron transfer step, but does not affect the follow-up reactivity of the electrogenerated ferrocenium cation, which leads eventually to the corresponding quinone methide, as reported in the absence of CD. Accordingly, the cytotoxicity of these beta-CD-encapsulated organometallic complexes in hormone-independent breast-cancer cells (MDA-MB231) were confirmed to be similar to those obtained in the absence of cyclodextrin.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Ciclodextrinas/farmacología , Compuestos Ferrosos/farmacología , Tamoxifeno/farmacología , Proliferación Celular/efectos de los fármacos , Ciclodextrinas/química , Diseño de Fármacos , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Electroquímica , Compuestos Ferrosos/síntesis química , Compuestos Ferrosos/química , Humanos , Metanol/química , Conformación Molecular , Estereoisomerismo , Tamoxifeno/química , Células Tumorales Cultivadas , Agua/químicaRESUMEN
We recently reported the dual (antihormonal and cytotoxic) functionality of ferrocifens, which are organometallic complexes derived from hydroxytamoxifen, the standard molecule in the treatment of hormone-dependent breast cancers. To test the hypothesis that the presence of a ferrocenyl substituent on molecules with an affinity for the estrogen receptor is sufficient to give them cytotoxic properties in vitro, we prepared complexes derived from estradiol with a ferrocenyl substituent at positions 7alpha and 17alpha. The complexes thus obtained retain a satisfactory level of affinity for the estrogen receptor (RBA values higher than 12 %). At low concentrations (0.1-1 microM) the complexes show an estrogenic effect in vitro equivalent to that of estradiol on hormone-dependent (MCF-7) breast cancer cells, and no cytotoxic effect on hormone-independent (MDA-MB-231) breast cancer cells. At high concentrations (up to 50 microM) the 17alpha-ethynylferrocenyl estradiol and 7alpha-ferrocenylmethylthio estradiol become cytotoxic (IC(50)=13.2 microM and 18.8 microM, respectively) while the 17alpha-ferrocenylestradiol remains non toxic. The low toxicity of these compounds support our hypothesis that electronic communication between the ferrocenyl and phenol moieties in the hydroxyferrocifens series is a key parameter in the generation of cytotoxic effects at submicromolar concentrations.
Asunto(s)
Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Estrógenos/química , Compuestos Ferrosos/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Electroquímica , Humanos , Metalocenos , Modelos Moleculares , Receptores de Estrógenos/metabolismoRESUMEN
A series of new derivatives of estradiol substituted at position 17alpha by various aryls has been synthesized. This was made possible by efficient activation methods for the addition of aryllithiums to the carbonyl group at position 17 of estrone by using tetramethylethylenediamine (TMEDA) or BF3 x OEt2. Their relative binding affinity (RBA) for the alpha and the beta forms of the estrogen receptor (ER) have been measured. All except one of the compounds synthesized had an RBA value of around 10 % which indicates a level of tolerance towards the bulky substituent at position 17. The lipophilicity values measured for these compounds are higher than that found for estradiol (E2). A study of their proliferative/antiproliferative effects was carried out on hormone-dependent (MCF7) and hormone-independent (MDA-MB231) breast cancer cell lines. It is interesting to note that all the compounds are estrogenic. The possibility of easily attaching an iodine at the end of a phenyl spacer opens up a route to new radiopharmaceuticals for use in radioimaging.