RESUMEN
A series of novel acridine N-acylhydrazone derivatives have been synthesized as potential topoisomerase I/II inhibitors, and their binding (calf thymus DNActDNA and human serum albuminHSA) and biological activities as potential anticancer agents on proliferation of A549 and CCD-18Co have been evaluated. The acridine-DNA complex 3b (-F) displayed the highest Kb value (Kb = 3.18 × 103 M−1). The HSA-derivatives interactions were studied by fluorescence quenching spectra. This method was used for the calculation of characteristic binding parameters. In the presence of warfarin, the binding constant values were found to decrease (KSV = 2.26 M−1, Kb = 2.54 M−1), suggesting that derivative 3a could bind to HSA at Sudlow site I. The effect of tested derivatives on metabolic activity of A549 cells evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT assay decreased as follows 3b(-F) > 3a(-H) > 3c(-Cl) > 3d(-Br). The derivatives 3c and 3d in vitro act as potential dual inhibitors of hTopo I and II with a partial effect on the metabolic activity of cancer cells A594. The acridine-benzohydrazides 3a and 3c reduced the clonogenic ability of A549 cells by 72% or 74%, respectively. The general results of the study suggest that the novel compounds show potential for future development as anticancer agents.
Asunto(s)
Antineoplásicos , Acridinas/química , Antineoplásicos/química , Sitios de Unión , Humanos , Sustancias Intercalantes , Albúmina Sérica Humana/química , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
A series of novel C4-C7-tethered biscoumarin derivatives (12a-e) linked through piperazine moiety was designed, synthesized, and evaluated biological/therapeutic potential. Biscoumarin 12d was found to be the most effective inhibitor of both acetylcholinesterase (AChE, IC50 = 6.30 µM) and butyrylcholinesterase (BChE, IC50 = 49 µM). Detailed molecular modelling studies compared the accommodation of ensaculin (well-established coumarin derivative tested in phase I of clinical trials) and 12d in the human recombinant AChE (hAChE) active site. The ability of novel compounds to cross the blood-brain barrier (BBB) was predicted with a positive outcome for compound 12e. The antiproliferative effects of newly synthesized biscoumarin derivatives were tested in vitro on human lung carcinoma cell line (A549) and normal colon fibroblast cell line (CCD-18Co). The effect of derivatives on cell proliferation was evaluated by MTT assay, quantification of cell numbers and viability, colony-forming assay, analysis of cell cycle distribution and mitotic activity. Intracellular localization of used derivatives in A549 cells was confirmed by confocal microscopy. Derivatives 12d and 12e showed significant antiproliferative activity in A549 cancer cells without a significant effect on normal CCD-18Co cells. The inhibition of hAChE/human recombinant BChE (hBChE), the antiproliferative activity on cancer cells, and the ability to cross the BBB suggest the high potential of biscoumarin derivatives. Beside the treatment of cancer, 12e might be applicable against disorders such as schizophrenia, and 12d could serve future development as therapeutic agents in the prevention and/or treatment of Alzheimer's disease.
Asunto(s)
Técnicas de Química Sintética , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Cumarinas/química , Cumarinas/farmacología , Modelos Moleculares , Células A549 , Enfermedad de Alzheimer/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores de la Colinesterasa/síntesis química , Cumarinas/síntesis química , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
A549 human lung carcinoma cell lines were treated with a series of new drugs with both tacrine and coumarin pharmacophores (derivatives 1a-2c) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. The ability of human topoisomerase I (hTOPI) and II to relax supercoiled plasmid DNA in the presence of various concentrations of the tacrine-coumarin hybrid molecules was studied with agarose gel electrophoresis. The biological activities of the derivatives were studied using MTT assays, clonogenic assays, cell cycle analysis and quantification of cell number and viability. The content and localization of the derivatives in the cells were analysed using flow cytometry and confocal microscopy. All of the studied compounds were found to have inhibited topoisomerase I activity completely. The effect of the tacrine-coumarin hybrid compounds on cancer cells is likely to be dependent on the length of the chain between the tacrine and coumarin moieties (1c, 1d = tacrine-(CH2)8-9-coumarin). The most active of the tested compounds, derivatives 1c and 1d, both display longer chains.
Asunto(s)
Antineoplásicos/farmacología , Cumarinas/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/antagonistas & inhibidores , Tacrina/farmacología , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/farmacología , Células A549 , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Cumarinas/química , ADN-Topoisomerasas de Tipo II/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Tacrina/química , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa II/química , Células Tumorales CultivadasRESUMEN
A series of new 3,6,9-trisubstituted acridine derivatives with fluorine substituents on phenyl ring were synthesized and their interaction with calf thymus DNA was investigated. Analysis using UV-Vis absorbance spectra provided valuable information about the formation of the acridine-DNA complex. In addition, compounds 8b and 8d were found to display an increased binding affinity (Kâ¯=â¯2.32 and 2.28â¯×â¯106 M-1, respectively). Topo I/II inhibition mode assays were also performed, and the results verify that the novel compounds display topoisomerase I and II inhibitory activity; compounds 8a, 8b and 8c completely inhibited topoisomerase I activity at a concentration of 60â¯×â¯10-6 M, but only compound 8d showed partial ability to inhibit topoisomerase II at concentrations of 30 and 50â¯×â¯10-6 M. The ability of the derivatives to impair cell proliferation was tested through an analysis of cell cycle distribution, quantification of cell number, viability studies, metabolic activity measurement and clonogenic assay. The content and localization of the derivatives in cells were analyzed using flow cytometry and fluorescence microscopy. The compounds 8b and 8d altered the physiochemical properties and improved antiproliferative activity in A549 human lung carcinoma cells (compound 8d displayed the highest level of activity, 4.25â¯×â¯10-6 M, after 48â¯h).
Asunto(s)
Acridinas/farmacología , Antineoplásicos/farmacología , ADN/efectos de los fármacos , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/farmacología , Células A549 , Acridinas/síntesis química , Acridinas/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Bovinos , Proliferación Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Halogenación , Humanos , Estructura Molecular , Relación Estructura-Actividad , Inhibidores de Topoisomerasa I/síntesis química , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/químicaRESUMEN
A combination of biochemical, biophysical and biological techniques was used to study calf thymus DNA interaction with newly synthesized 7-MEOTA-tacrine thiourea 12-17 and urea heterodimers 18-22, and to measure interference with type I and II topoisomerases. Their biological profile was also inspected in vitro on the HL-60 cell line using different flow cytometric techniques (cell cycle distribution, detection of mitochondrial membrane potential dissipation, and analysis of metabolic activity/viability). The compounds exhibited a profound inhibitory effect on topoisomerase activity (e.g. compound 22 inhibited type I topoisomerase at 1 µM concentration). The treatment of HL-60 cells with the studied compounds showed inhibition of cell growth especially with hybrids containing thiourea (14-17) and urea moieties (21 and 22). Moreover, treatment of human dermal fibroblasts with the studied compounds did not indicate significant cytotoxicity. The observed results suggest beneficial selectivity of the heterodimers as potential drugs to target cancer cells.
Asunto(s)
Acridinas/farmacología , Antineoplásicos/farmacología , Tacrina/farmacología , Tiourea/farmacología , Células A549 , Acridinas/síntesis química , Acridinas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Fibroblastos/efectos de los fármacos , Células HL-60 , Humanos , Relación Estructura-Actividad , Tacrina/química , Tiourea/químicaRESUMEN
Photodynamic therapy with hypericin (HY-PDT) and hyperforin (HP) could be treatment modalities for colorectal cancer (CRC), but evidence of their effect on angiogenic factors in CRC is missing. Convenient experimental model utilization is essential for angiogenesis research. Therefore, not only 2D cell models, but also 3D cell models and micro-tumors were used and compared. The micro-tumor extent and interconnection with the chorioallantoic membrane (CAM) was determined by histological analyses. The presence of proliferating cells and HY penetration into the tumor mass were detected by fluorescence microscopy. The metabolic activity status was assessed by an colorimetric assay for assessing cell metabolic activity (MTT assay) and HY accumulation was determined by flow cytometry. Pro-angiogenic factor expression was determined by Western blot and quantitative real-time polymerase chain reaction (RT-qPCR). We confirmed the cytotoxic effect of HY-PDT and HP and showed that their effect is influenced by structural characteristics of the experimental model. We have pioneered a method for analyzing the effect of HP and cellular targeted HY-PDT on pro-angiogenic factor expression in CRC micro-tumors. Despite the inhibitory effect of HY-PDT and HP on CRC, the increased expression of some pro-angiogenic factors was observed. We also showed that CRC experimental micro-tumors created on quail CAM could be utilized for analyses of gene and protein expression.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neovascularización Patológica/metabolismo , Perileno/análogos & derivados , Floroglucinol/análogos & derivados , Fotoquimioterapia , Terpenos/farmacología , Inductores de la Angiogénesis/química , Animales , Antracenos , Biomarcadores , Línea Celular Tumoral , Embrión de Pollo , Membrana Corioalantoides/patología , Neoplasias Colorrectales/terapia , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Neovascularización Patológica/terapia , Perileno/química , Perileno/farmacología , Floroglucinol/química , Floroglucinol/farmacología , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Terpenos/químicaRESUMEN
Cancer cells disseminate to other parts of the body during metastasis through the process of intravasation. The hypericin and hyperforin effect has been described to understand the signal mechanisms that stimulate or stunt cancer cell sprouting to metastasis on colon adenocarcinoma cells HT-29 and its resistant form HT-29-OxR. We focused on the key points of adhesion proteins (cadherin, integrin, selectin and syndecan) and also proteins participating in or contributing to the process of cancer cell migration and adhesion through genes expression and proteins levels. Treatment effects were identified as a consequence of decreased cell adhesion, changes of expression in the adhesive proteins as well as basal membrane degradation associated with changes in the expression of matrix proteinases and in their activity. Finally, the cells affected by hypericin or hyperforin were evaluated by monitoring the cancer cell adhesion properties and proliferation processes. Supplementary Fig. (Supplemental digital content 1, http://links.lww.com/ACD/A267).
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Oxaliplatino/farmacología , Perileno/análogos & derivados , Floroglucinol/análogos & derivados , Terpenos/farmacología , Adenocarcinoma/patología , Antracenos , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Neoplasias del Colon/patología , Resistencia a Antineoplásicos , Células HT29 , Humanos , Metástasis de la Neoplasia/prevención & control , Oxaliplatino/administración & dosificación , Perileno/administración & dosificación , Perileno/farmacología , Floroglucinol/administración & dosificación , Floroglucinol/farmacología , Terpenos/administración & dosificaciónRESUMEN
Photodynamic therapy (PDT) represents a rapidly developing alternative treatment for various types of cancers. Although considered highly effective, cancer cells can exploit various mechanisms, including the upregulation of apoptosis inhibitors, to overcome the cytotoxic effect of PDT. Survivin, a member of the inhibitor of apoptosis protein family, is known to play a critical role in cancer progression and therapeutic resistance and therefore represents a potential therapeutic target. The aim of this study was to investigate whether YM155, a small molecule inhibitor of survivin expression, can potentiate the cytotoxic effect of hypericin-mediated PDT (HY-PDT). Accordingly, two cell lines resistant to HY-PDT, HT-29 (colorectal adenocarcinoma) and A549 (lung adenocarcinoma), were treated either with HY-PDT alone or in combination with YM155. The efficacy of different treatment regimens was assessed by MTT assay, flow cytometry analysis of metabolic activity, viability, phosphatidylserine externalisation, mitochondrial membrane potential and caspase-3 activity and immunoblotting for the cleavage of poly (ADP-ribose) polymerase (PARP). Here we show for the first time that the repression of survivin expression by YM155 is effective in sensitizing HT-29 and A549 cells to HY-PDT, as measured by the decrease in cell viability and induction of apoptosis. Combined treatment with hypericin and YM155 led to a more severe dissipation of the mitochondrial membrane potential and caused an increase in caspase-3 activation and subsequent PARP cleavage. Our results demonstrate that the repression of survivin expression by YM155 potentially represents a novel alternative strategy to increase the efficacy of HY-PDT in cancer cells that are otherwise weakly responsive or non-responsive to treatment.
Asunto(s)
Antineoplásicos/farmacología , Imidazoles/farmacología , Naftoquinonas/farmacología , Perileno/análogos & derivados , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma del Pulmón , Antracenos , Autofagia/efectos de los fármacos , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Resistencia a Antineoplásicos , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Perileno/farmacología , Fotoquimioterapia/métodos , SurvivinRESUMEN
This study examines the binding properties of a series of newly synthetized tacrine derivatives 1-4 and their anticancer effects. Spectroscopic techniques (UV-Vis, fluorescence spectroscopy, thermal denaturation, and linear spectropolarimetry) and viscometry were used to study DNA binding properties and to determine the types of DNA interaction with the studied derivatives. The binding constants for the complexes with DNA were obtained using UV-Vis spectroscopic titrations (K = 1.6 × 10(4)-4.0 × 10(5) M(-1)) and electrophoretic methods were used to determine the effect of the derivatives on topoisomerase I and II activity. Monotacrine derivative 1 showed evidence of topoisomerase Irelaxation activity at a concentration of 30 × 10(-6) M, while bistacrine derivatives 2-4 produced a complete inhibition of topoisomerase Iat a concentration of 5 × 10(-6) M. The biological activities of the derivatives were studied using MTT-assay and flow cytometric methods (detection of mitochondrial membrane potential and measurement of cell viability) following incubation of 24 and 48 h with human leukemic cancer cell line HL60. The ability of the derivatives to impair cell proliferation was also tested through the analysis of cell cycle distribution.
Asunto(s)
Antineoplásicos/farmacología , Tacrina/farmacología , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/farmacología , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN/metabolismo , ADN-Topoisomerasas/metabolismo , Células HL-60 , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Tacrina/química , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa II/químicaRESUMEN
5-Bromo-2'-deoxyuridine (BrdU) is a marker that is widely used to label S-phase cells in neurobiological research in most common doses 50 or 100 mg/kg per single intraperitoneal (i.p.) injection. However, the important data regarding its pharmacokinetics in rodents are still missing. The aim of our study was to investigate the BrdU level in serum after a single i.p. injection to adult rats (doses: 50 or 100 mg/kg) and adult mice (50 mg/kg). The animals were killed at selected time-points after the BrdU injection, and proliferating tumour cells (cell lines HCT-116 and HL-60) were co-cultivated with isolated blood sera. BrdU incorporated in the DNA of the S-phase tumour cells was stained with an anti-BrdU antibody and analysed using flow cytometry. In rats, the efficacies of BrdU labelling of S-phase cells in both in vitro and in vivo conditions were compared in the 50 and 100 mg/kg groups. According to our results, BrdU was in saturated concentration to label almost all S-phase cells for 60 min in both doses and was detectable in blood serum until 120 min after the single i.p. injection. However, the 100 mg/kg dose of BrdU did not provide a prolonged staining period to offset the potentially higher toxicity in comparison with the 50 mg/kg dose. In mice, due to their faster metabolism, the concentration of BrdU in blood serum was sufficient to label the whole population of S-phase cells for only 15 min after the i.p. injection, then dropped rapidly.
Asunto(s)
Bromodesoxiuridina/farmacocinética , Citometría de Flujo , Animales , Bromodesoxiuridina/administración & dosificación , Bromodesoxiuridina/sangre , Relación Dosis-Respuesta a Droga , Células HCT116 , Humanos , Inmunohistoquímica , Inyecciones Intraperitoneales , Ratones , RatasRESUMEN
A novel series of trisubstituted acridines were synthesized with the aim of mimicking the effects of BRACO19. These compounds were synthesized by modifying the molecular structure of BRACO19 at positions 3 and 6 with heteroacyclic moieties. All of the derivatives presented in the study exhibited stabilizing effects on the human telomeric DNA quadruplex. UV-vis spectroscopy, circular dichroism, linear dichroism and viscosimetry were used in order to study the nature of the DNA binding in more detail. The results show that all of the novel derivatives were able to fold the single-stranded DNA sequences into antiparallel G-quadruplex structures, with derivative 15 exhibiting the highest stabilizing capability. Cell cycle analysis revealed that a primary trend of the "braco"-like derivatives was to arrest the cells in the S- and G2M-phases of the cell cycle within the first 72h, with derivative 13 and BRACO19 proving particularly effective in suppressing cell proliferation. All studies derivatives were less toxic to human fibroblast cell line in comparison with HT 29 cancer cell line.
Asunto(s)
Acridinas/química , Acridinas/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , ADN/metabolismo , G-Cuádruplex/efectos de los fármacos , Animales , Bovinos , Ciclo Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN/química , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismoRESUMEN
The presence of key hypoxia regulators, namely, hypoxia-inducible factor (HIF)-1α or HIF-2α, in tumors is associated with poor patient prognosis. Hypoxia massively activates several genes, including the one encoding the BCRP transporter that proffers multidrug resistance to cancer cells through the xenobiotic efflux and is a determinant of the side population (SP) associated with cancer stem-like phenotypes. As natural medicine comes to the fore, it is instinctive to look for natural agents possessing powerful features against cancer resistance. Hypericin, a pleiotropic agent found in Hypericum plants, is a good example as it is a BCRP substrate and potential inhibitor, and an SP and HIF modulator. Here, we showed that hypericin efficiently accumulated in hypoxic cancer cells, degraded HIF-1/2α, and decreased BCRP efflux together with hypoxia, thus diminishing the SP population. On the contrary, this seemingly favorable result was accompanied by the stimulated migration of this minor population that preserved the SP phenotype. Because hypoxia unexpectedly decreased the BCRP level and SP fraction, we compared the SP and non-SP proteomes and their changes under hypoxia in the A549 cell line. We identified differences among protein groups connected to the epithelial-mesenchymal transition, although major changes were related to hypoxia, as the upregulation of many proteins, including serpin E1, PLOD2 and LOXL2, that ultimately contribute to the initiation of the metastatic cascade was detected. Altogether, this study helps in clarifying the innate and hypoxia-triggered resistance of cancer cells and highlights the ambivalent role of natural agents in the biology of these cells.
Asunto(s)
Neoplasias , Células de Población Lateral , Humanos , Células de Población Lateral/patología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Hipoxia , Neoplasias/metabolismo , Hipoxia de la Célula , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Línea Celular Tumoral , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación Neoplásica de la Expresión GénicaRESUMEN
It is more than sixty years since the era of modern photodynamic therapy (PDT) for cancer began. Enhanced selectivity for malignant cells with a reduced selectivity for non-malignant cells and good biocompatibility along with the limited occurrence of side effects are considered to be the most significant advantages of PDT in comparison with conventional therapeutic approaches, e.g., chemotherapy. The phenomenon of multidrug resistance, which is associated with drug efflux transporters, was originally identified in relation to the application of chemotherapy. Unfortunately, over the last thirty years, numerous papers have shown that many photosensitizers are the substrates of efflux transporters, significantly restricting the effectiveness of PDT. The concept of a dynamic nanoplatform offers a possible solution to minimize the multidrug resistance effect in cells affected by PDT. Indeed, recent findings have shown that the utilization of nanoparticles could significantly enhance the therapeutic efficacy of PDT. Additionally, multifunctional nanoplatforms could induce the synergistic effect of combined treatment regimens, such as PDT with chemotherapy. Moreover, the surface modifications that are associated with nanoparticle functionalization significantly improve the target potential of PDT or chemo-PDT in multidrug resistant and cancer stem cells.
RESUMEN
Preferential uptake of photosensitizer by tumour tissue is an elementary prerequisite of effective and successful photodynamic therapy (PDT). Therefore intracellular concentration of photosensitizer is one of the limiting factors affecting PDT efficiency. Hypericin (HY) has found applications in photodynamic diagnostics solely due to its high specificity for tumour cells and tissues. However, here we suggest that not only HY uptake, but importantly also the cell ability to manage oxidative stress induced by HY-PDT can be important decisive factors finally affecting the cell death response. We showed that despite the higher accumulation of HY in FHC human fetal colon epithelial cells compared to HT-29 colon adenocarcinoma cells, the cytotoxic effects of this photosensitizer were more pronounced in the latter cell line, and this was associated with enhanced accumulation of HY-PDT-induced reactive oxygen species (ROS).
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Células Epiteliales/efectos de los fármacos , Perileno/análogos & derivados , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Antracenos , Línea Celular , Colon/citología , Células Epiteliales/efectos de la radiación , Feto , Humanos , Microscopía Fluorescente , Perileno/uso terapéutico , Perileno/toxicidad , Fármacos Fotosensibilizantes/toxicidad , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Several photodynamically-active substances and farnesyltransferase inhibitors are currently being investigated as promising anticancer drugs. In this study, the combined effect of hypericin (the photodynamically-active pigment from Hypericum perforatum) and selective farnesyltransferase inhibitor manumycin (manumycin A; the selective farnesyltransferase inhibitor from Streptomyces parvulus) on HT-29 adenocarcinoma cells was examined. We found that the combination treatment of cells with photoactivated hypericin and manumycin resulted in enhanced antiproliferative and apoptotic response compared to the effect of single treatments. This was associated with increased suppression of clonogenic growth, S phase cell cycle arrest, elevated caspase-3/7 activity and time-dependent total cleavage of procaspase-3 and lamin B, cleavage of p21Bax into p18Bax and massive PARP cleavage. Moreover, we found that the apoptosis-inducing factor is implicated in signaling events triggered by photoactivated hypericin. Our results showed the relocalization of apoptosis-inducing factor (AIF) to the nuclei after hypericin treatment. In addition, we discovered that not only manumycin but also photoactivated hypericin induced the reduction of total Ras protein level. Manumycin decreased the amount of farnesylated Ras, and the combination treatment decreased the amount of both farnesylated and non-farnesylated Ras protein more dramatically. The present findings indicate that the inhibition of Ras processing may be the determining factor for enhancing the antiproliferative and apoptotic effects of combination treatment on HT-29 cells.
Asunto(s)
Adenocarcinoma/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Perileno/análogos & derivados , Polienos/farmacología , Alcamidas Poliinsaturadas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Antracenos , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/genética , Caspasa 7/metabolismo , Inhibidores Enzimáticos/farmacología , Células HT29 , Humanos , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Perileno/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Skyrin (SKR) is a plant bisanthraquinone secondary metabolite from the Hypericum genus with potential use in anticancer therapy. However, its effect and mechanism of action are still unknown. The negative effect of SKR on HCT 116 and HT-29 cancer cell lines in hypoxic and normoxic conditions was observed. HCT 116 cells were more responsive to SKR treatment as demonstrated by decreased metabolic activity, cellularity and accumulation of cells in the G1 phase. Moreover, an increasing number of apoptotic cells was observed after treatment with SKR. Based on the LC-MS comparative proteomic data from hypoxia and normoxia (data are available via ProteomeXchange with the identifier PXD019995), SKR significantly upregulated Death receptor 5 (DR5), which was confirmed by real-time qualitative PCR (RT-qPCR). Furthermore, multiple changes in the Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-activated cascade were observed. Moreover, the reversion of TRAIL resistance was observed in HCT 116, HT-29 and SW620 cell lines, even in hypoxia, which was linked to the upregulation of DR5. In conclusion, our results propose the use of SKR as a prospective anticancer drug, particularly as an adjuvant to TRAIL-targeting treatment to reverse TRAIL resistance in hypoxia.
RESUMEN
Since many studies have suggested the impact of dietary polyunsaturated fatty acids on cancer progression and prognosis, there is an assumption of possible pre-sensitizing effects of their application in combined treatment. The present work evaluates modulation of photodynamic therapy (PDT) with hypericin by pre-treatment with n-3 and n-6 fatty acids in HT-29 and HeLa tumour cells. We observed stimulation of cytotoxic effects by docosahexaenoic acid (n-3) and arachidonic acid (n-6) in several stages of action in both cell lines. Treatment with either fatty acids or photodynamic therapy alone induced apoptosis in a dose- and time-dependent manner; however the effect was even more striking in mutual combination applied as pre-treatment with fatty acids prior to photodynamic therapy. Moreover, the combination also induced changes in membrane lipid composition leading to alteration in cell membrane fluidity. Increased toxicity of combined treatment was also confirmed by the presence of oxidative stress demonstrated by ROS production, RNS accumulation and increased presence of lipoperoxides. In conclusion, we suggest that pre-treatment with polyunsaturated fatty acids may contribute to cytotoxic effects induced by photodynamic therapy with hypericin.
Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis , Ácidos Grasos Insaturados/toxicidad , Estrés Oxidativo , Perileno/análogos & derivados , Antracenos , Ácido Araquidónico/toxicidad , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Ácidos Docosahexaenoicos/toxicidad , Células HT29 , Humanos , Fluidez de la Membrana , Perileno/uso terapéutico , FotoquimioterapiaRESUMEN
The relevance of experimentally gained information represents a long-term debating issue in the field of molecular biology research. The loss of original conditions in the in vitro environment affects various biological mechanisms and cellular interactions. Consequently, some biochemical mechanisms are lost or critically altered. Analyses in these modified conditions could, therefore, distort the relevancy of experimentally gained information. In some cases, the similarities with original conditions are so small that utilization of simpler in vitro models seems impossible, or could occur in a very limited way. To conclude, the study of more complex phenomena places higher demands on the complexity of the experimental model. The latest information highlights the fact that the tumor angiogenesis mechanism has very complex features. This complexity can be associated with a wide range of angiogenic factors expressed by a variety of malignant and non-malignant cells. Our article summarizes the results from various experimental models that were utilized to analyze a photodynamic therapy effect on tumor angiogenic mechanisms. Additionally, based on the latest information, we present the most important attributes and limitations of utilized experimental models. We also evaluate the essential problems associated with angiogenic mechanism induction after photodynamic therapy application.
RESUMEN
The use of natural products as chemotherapeutic agents and tools for manipulation of apoptosis represent an attractive therapeutic concept. In this study, we investigated the anticancer activities of a combination of two natural compounds with different origin, hypericin (plant product) in its photoactive state and Manumycin A (yeast product) and explored the underlying mechanisms of their pro-apoptotic action using an oxaliplatin-resistant variant of human colon adenocarcinoma cell line HT-29-OxR as the experimental model. CCK-8 assay was performed to evaluate the cytotoxicity of the drugs. CalcuSyn software was used to identify the type of interaction between the two agents. BrdU incorporation assay and colony forming assay were performed to study the short- and long-term proliferation of cells. To evaluate the ability of the drug combination to induce apoptosis, PARP p85 fragment was detected using the ELISA method. Changes in apoptosis-related proteins were examined by immunoassays. Our results showed that a synergistic combination of photoactive hypericin and Manumycin A decreased viability, inhibited both short- and long-term cell proliferation, decreased levels of IAPs proteins (cIAP1, cIAP2, XIAP and survivin), induced an apoptotic PARP cleavage associated with decline in procaspase-3 level, promoted phagocytosis of cancer cells, and restored chemosensitivity to oxaliplatin.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Perileno/análogos & derivados , Polienos/farmacología , Alcamidas Poliinsaturadas/farmacología , Antracenos , Antineoplásicos/efectos de la radiación , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Luz , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Oxaliplatino/farmacología , Perileno/farmacología , Perileno/efectos de la radiación , Fagocitosis/efectos de los fármacosRESUMEN
Hypericin (HY) is a naphthodianthrone that naturally occurs in Hypericum perforatum L. It is a promising photosensitiser used in photodynamic therapy for and diagnosis of oncological diseases. However, its hydrophobic character is an obstacle that has prevented its efficient use. The commonly used solvent, dimethyl sulfoxide (DMSO), is a controversial constituent of HY formulations and its use has been rejected by many researchers studying HY both in vitro and in vivo. In this study, we propose the utilisation of hydrotropy to solubilise HY in an aqueous environment. Cromolyn (DSCG) is a non-toxic, well-tolerated, antiallergic drug that has been employed in clinical practice since 1970, and in aqueous solution it acts as a hydrotrope. At a molecular ratio of 1:12,000 HY to DSCG, the compound is able to solubilise HY in aqueous environment. In an HT-29 cell suspension, DSCG (1.8â¯mmolâ¯L-1) considerably enhances the interaction between HY (150â¯nmolâ¯L-1) and HT-29 cells, which leads to an HY fluorescence emission increase with a half-time approximately 2â¯min compared to 29â¯min for samples that lack DSCG. Studies using HT-29 adenocarcinoma cells showed that DSCG at a given concentration significantly improved accumulation of HY within cells compared to DMSO (p < 0.05) despite the relative resistance of the HT-29 cell line to HY-PDT. Though no significant difference between total reactive oxygen species production was observed for photoactivated HY dissolved in DMSO and DSCG, significant singlet oxygen generation by photoactivated HY dissolved in a DSCG-containing water solution at the studied molecular ratio was confirmed. We also clarified that DSCG does not act as a scavenger of ABTS and galvinoxyl free radicals. The results from an MTT assay showed that DSCG also significantly enhanced the cytotoxicity of photoactivated HY compared to DMSO (p < 0.05). This study has demonstrated the ability of DSCG to act as a solvent of HY and enhance the effectiveness of HY-PDT compared to the commonly used organic solvent, DMSO.