RESUMEN
PURPOSE: Although fenretinide (4-HPR) has been studied in breast cancer and in neuroblastoma, little is known regarding its activity in pancreatic cancer, a neoplasm for which there are few therapeutic options. Since pancreatic cancer cells are susceptible to reactive oxygen species (ROS) and ceramide, two hallmarks of 4-HPR cytotoxicity, we investigated the effect of 4-HPR on human pancreatic cancer cells. METHODS: Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were treated with 4-HPR, followed by measurement of viability, proliferation, ROS and ceramide production, and Western blotting. RESULTS: At the measured IC(50) of 10 µM, 4-HPR led to a 44-68% reduction in [(3)H]thymidine incorporation, a >3-fold increase in de novo ceramide levels, a 2.7-fold increase in ROS, and minor increases in markers of apoptosis. 4-HPR induced a robust, sustained increase in LC3 II expression and enhanced formation of acridine orange-stained acidic vesicles that are markers of autophagy. In addition, sustained, dose-dependent increases in JNK and p38 phosphorylation and decreased ERK phosphorylation were observed following treatment. Pretreatment with vitamin E, a ROS scavenger, and 3-methyladenine, an autophagy inhibitor, individually led to decreased sensitivity to 4-HPR; however, the de novo ceramide inhibitor myriocin had no effect. CONCLUSIONS: These data show that 4-HPR triggers pancreatic cancer cell death by apoptosis and autophagy and that sensitivity appears to be mediated by ROS and not ceramide. This study is the first to characterize the response of human pancreatic cancer cells to 4-HPR and opens the door to investigations into this compound in pancreatic adenocarcinomas.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma/tratamiento farmacológico , Fenretinida/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Autofagia/efectos de los fármacos , Biomarcadores/metabolismo , Carcinoma/metabolismo , Carcinoma/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ceramidas/antagonistas & inhibidores , Ceramidas/biosíntesis , Fenretinida/antagonistas & inhibidores , Depuradores de Radicales Libres/farmacología , Humanos , Concentración 50 Inhibidora , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismoRESUMEN
N-(4-hydroxyphenyl)retinamide (4HPR) is a synthetic retinoid that has been tested in clinical trials as a cancer chemopreventive drug. 4HPR is cytotoxic to cancer cells but the underlying molecular mechanisms are at present only partially understood. Here we demonstrate that in the human cervical cancer cell line HeLa and the human leukemia cell line HL-60, 4HPR caused rapid, Reactive Oxygen Species (ROS)-dependent activation of the Unfolded Protein Response (UPR). In HeLa cells, 4HPR was shown to induce cell death and activation of procaspases. These effects of 4HPR could be abolished by the over-expression of dominant negative mutants of PERK or eIF2 alpha. HeLa cells incubated with 4HPR were found to form autophagosomes that were also mediated by the PERK/eIF2 alpha pathway. While 4HPR-induced cell death could be significantly prevented by the presence of specific caspase inhibitors, 3-methyladenine (3-MA) that inhibits autophagosome formation enhanced 4HPR-induced cell death. Examination of individual 4HPR-treated HeLa cells revealed that those without the development of autophagosomes hence exhibiting an incomplete UPR were caspase-active and were not viable, while those with autophagosomes were caspase-inactive and retained cell viability. Our data suggest that the PERK/eIF2 alpha pathway is essential for the cytotoxicity of 4HPR that targets on cancer cells with malfunctional UPR.
Asunto(s)
Anticarcinógenos/toxicidad , Factor 2 Eucariótico de Iniciación/metabolismo , Fenretinida/toxicidad , Transducción de Señal , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 6/metabolismo , Anticarcinógenos/antagonistas & inhibidores , Apoptosis , Autofagia , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Precursores Enzimáticos/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Fenretinida/antagonistas & inhibidores , Células HL-60 , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Mutación , Fagosomas/metabolismo , Pliegue de Proteína , Empalme del ARN , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , eIF-2 Quinasa/genéticaRESUMEN
The question whether chemotherapy-induced autophagy is causative to the demise of the cells or a part of the survival mechanism activated during cellular distress is unclear. Others and we have previously demonstrated apoptosis-inducing capacity of N-(4-hydroxyphenyl)retinamide (4-HPR) in malignant glioma cells. We provide evidences of 4-HPR-induced autophagy at a lower concentration (5 microM). Suboptimal dose of 4-HPR treatment of malignant glioma cell lines increased G(2)/M arrest, whereas cell accumulated in S phase at a higher concentration. 4-HPR-induced autophagy was associated with acidic vacuole [acidic vesicular organelle (AVO)] formation and recruitment of microtubule-associated protein light chain 3 (LC3). At a higher concentration of 10 microM of 4-HPR, glioma cells undergoing apoptosis manifested autophagic features indicated by autophagosome formation, AVO development and LC3 localization. Autophagy inhibition at an early stage by 3-methyl adenine inhibited the AVO formation and LC3 localization with an enhancement in cell death. Bafilomycin A1, a specific inhibitor of vacuolar type Hthorn-ATPase also prevented AVO formation without effecting LC-3 localization pattern and also enhanced the extent of 4-HPR-induced cell death. 4-HPR activated c-jun and P38(MAPK) at both 5 and 10 microM concentrations, whereas increased activation of extracellular signal-regulated kinase 1/2 and NF-kappaB was seen only at lower dose. Inhibiting phosphoinositide 3-kinase and mitogen-activated protein kinases pathways modulated 4-HPR-induced cell death. This is the first report that provides evidences that besides apoptosis induction 4-HPR can also induce autophagy. These results indicate that 4-HPR-induced autophagy in glioma cell may provide survival advantage and inhibition of autophagy may enhance the cytotoxicity to 4-HPR.
Asunto(s)
Autofagia/efectos de los fármacos , Neoplasias Encefálicas/patología , Muerte Celular/efectos de los fármacos , Fenretinida/antagonistas & inhibidores , Glioma/patología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Fenretinida/farmacología , Citometría de Flujo , Humanos , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Proteínas Quinasas/metabolismoRESUMEN
Butyrates and retinoids are promising antineoplastic agents. Here we analyzed effects of sodium butyrate and N-(4-hydroxyphenyl)-retinamide (4-HPR) on prostate cancer cells as monotherapy or in combination in vitro and in vivo. Sodium butyrate and 4-HPR induced concentration-dependent growth inhibition in prostate cancer cells in vitro. The isobologram analysis revealed that sodium butyrate and 4-HPR administered together antagonize effects of each other. For the in vivo studies, a water-soluble complex (4-HPR with a cyclodextrin) was created. A single dose of sodium butyrate and 4-HPR showed a peak level in chicken plasma within 30 minutes. Both compounds induced inhibition of proliferation and apoptosis in xenografts of the chicken chorioallantoic membrane. Analysis of the cytotoxic effects of the drugs used in combination demonstrated an antagonistic effect on inhibition of proliferation and on induction of apoptosis. Prolonged jun N-terminal kinase phosphorylation induced by sodium butyrate and 4-HPR was strongly attenuated when both compounds were used in combination. Both compounds induced inhibition of NF-kappaB. This effect was strongly antagonized in LNCaP cells when the compounds were used in combination. These results indicate that combinational therapies have to be carefully investigated due to potential antagonistic effects in the clinical setting despite promising results of a monotherapy.
Asunto(s)
Butiratos/farmacología , Fenretinida/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Butiratos/antagonistas & inhibidores , Butiratos/farmacocinética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Pollos , Membrana Corioalantoides/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fenretinida/antagonistas & inhibidores , Fenretinida/farmacocinética , Humanos , Masculino , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Trasplante de Neoplasias , Neoplasias de la Próstata/patología , Trasplante HeterólogoRESUMEN
Approximately 30-40% of estrogen receptor alpha (ERalpha)-positive breast tumors express high levels of the cyclooxygenase-2 (COX-2) protein, and these high levels have been associated with a poorer prognosis in breast cancer patients. We speculate that high levels of COX-2 induce drug resistance in ERalpha-positive breast tumors, thus reducing the survival rate of patients with such tumors. Human breast cancer cell lines that express high levels of COX-2 are generally ERalpha negative. To determine whether COX-2 induces drug resistance, plasmids encoding the COX-2 gene were stably transfected into ERalpha-positive MCF-7 human breast cancer cells (MCF-7/COX-2). MCF-7/COX-2 cells were resistant to the selective estrogen receptor modulator tamoxifen but not to its analog, raloxifene. MCF-7/COX-2 cells were also resistant to the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) but not to its analog, all-trans retinoic acid. In contrast, the sensitivities of MCF-7/COX-2 cells to doxorubicin and paclitaxel were similar to those of the parental MCF-7 cells. We then determined which COX-2 product, prostaglandin E2 (PGE2) or prostaglandin F2alpha is involved in the COX-2-mediated drug resistance. PGE2, but not PGF2alpha, blocked the antiproliferative effects of tamoxifen and 4-HPR. Agonists that activate PGE2 receptors and their downstream kinase effectors, protein kinases A and C, also blocked the growth inhibitory effects of these drugs. Increased levels of Bcl-2 and Bcl-XL proteins have been reported in mammary tumors of COX-2 transgenic mice and in human colon cancer cell lines that have high levels of COX-2. However, we did not observe any changes in Bcl-2, Bcl-XL, or Bax expression induced by COX-2 or PGE2. Here we report the novel findings that COX-2 uses PGE2 to stimulate the activities of protein kinases A and C to induce selectively tamoxifen and 4-HPR resistance in ERalpha-positive breast cancer cells.
Asunto(s)
Antineoplásicos/metabolismo , Ciclooxigenasa 2/metabolismo , Fenretinida/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/metabolismo , Tamoxifeno/antagonistas & inhibidores , Neoplasias de la Mama/patología , Línea Celular Tumoral , Células Clonales , Resistencia a Antineoplásicos , Femenino , Humanos , Concentración 50 InhibidoraRESUMEN
The inhibitory effects of N-(4-hydroxyphenyl)retinamide (4HPR) on the process of carcinogenesis are not fully understood and may result from its ability to induce apoptosis in transformed cells. This study investigated the apoptotic properties of 4HPR in four human cutaneous squamous cell carcinoma cell lines. Apoptosis induction, detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling method, occurred in a dose- and time-dependent fashion after treatment with 4HPR. 4HPR promoted reactive oxygen species (ROS) determined by oxidation of 2',7'-dichlorofluorescin. 4HPR-induced ROS, and apoptosis could be inhibited by L-ascorbic acid. Rhodamine 123 retention revealed that 4HPR treatment promoted a gradual dissipation of mitochondrial inner transmembrane potential, and this could be inhibited by L-ascorbic acid, implying that mitochondrial permeability transition was involved in apoptosis induction. Cyclosporin A and bongkrekic acid inhibited dissipation of mitochondrial inner transmembrane potential, ROS production, and DNA fragmentation after exposure to 4HPR, demonstrating that mitochondrial permeability transition was a central coordinating feature of 4HPR-induced apoptosis.
Asunto(s)
Anticarcinógenos/farmacología , Apoptosis/fisiología , Fenretinida/farmacología , Membranas Intracelulares/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Anticarcinógenos/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Ácido Ascórbico/farmacología , Carcinoma de Células Escamosas/fisiopatología , División Celular/efectos de los fármacos , Ciclosporina/farmacología , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/farmacología , Fenretinida/antagonistas & inhibidores , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Membranas Intracelulares/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mitocondrias/fisiología , Oxidación-Reducción , Permeabilidad , Especies Reactivas de Oxígeno/metabolismo , Rodamina 123/metabolismo , Neoplasias Cutáneas/fisiopatología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The induction of apoptosis by the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) has been documented in vitro in various cancer types. A role for reactive oxygen species (ROS) in apoptosis induced by 4HPR in some cancer cells has been demonstrated recently. We studied five different human head and neck and five lung cancer cell lines to determine whether the ROS play a general role in 4HPR-induced apoptosis. We found that 4HPR induced apoptosis in all of the cell lines; however, this effect was blocked by antioxidants in only 2 of the 10 cell lines. 4HPR induced a greater than 4-fold increase in the generation of intracellular ROS in these two cell lines compared with a much lower effect in other cell lines. Furthermore, these two cell lines were most sensitive to the induction of apoptosis by 4HPR. The level of the cellular antioxidant thiol and superoxide dismutase activity were relatively lower in cells, which responded to 4HPR with a high level of ROS generation. These results indicate that although ROS can mediate 4HPR-induced apoptosis in some cells, which may have a low endogenous cellular antioxidant levels, other mechanisms exist for 4HPR-induced apoptosis. One such mechanism may involve retinoic acid receptors (RARs) because an RAR antagonist was able to block partially 4HPR-induced apoptosis. In conclusion, 4HPR-induced apoptosis involves at least three different mechanisms, which are complex and can overlap in the same cell line: (a) one mechanism involving 4HPR-induced ROS; (b) one involving RARs; and (c) at least one that does not involve ROS or RARs and remains unclear.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/patología , Fenretinida/farmacología , Neoplasias de Cabeza y Cuello/patología , Neoplasias Pulmonares/patología , Antineoplásicos/antagonistas & inhibidores , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Fenretinida/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Naftalenos/farmacología , Proteínas de Neoplasias/análisis , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno , Receptores de Ácido Retinoico/efectos de los fármacos , Receptores de Ácido Retinoico/fisiología , Compuestos de Sulfhidrilo/análisis , Superóxido Dismutasa/análisis , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
BACKGROUND: The inhibitory effects of N-(4-hydroxyphenyl)retinamide (4HPR) on tumorigenesis and tumor growth may result from its ability to induce apoptosis (programmed cell death). Since antioxidants inhibit 4HPR-induced apoptosis, experiments were planned to determine whether the levels of reactive oxygen species increase in cells undergoing apoptosis after exposure to 4HPR. METHODS: Cells of the human cervical carcinoma cell line C33A and normal human cervical epithelial cells were treated with 4HPR and analyzed for survival, induction of apoptosis, generation of reactive oxygen species, and expression of the apoptosis-related proteins Bcl-2 and Bax. RESULTS: Treatment with 4HPR decreased C33A cell number by inducing apoptosis in a time- and dose-dependent fashion. DNA fragmentation typical of apoptosis was observed in cells exposed to 4HPR at concentrations of 3 microM or higher for 6-24 hours. The generation of reactive oxygen species was enhanced by 1.85-fold to 4.5-fold after a 1.5-hour treatment with 0.4-10 microM 4HPR. Pyrrolidine dithiocarbamate, an oxygen radical scavenger, suppressed the rate of generation of reactive oxygen species and inhibited 4HPR-induced apoptosis. 4HPR failed to modulate cellular levels of the Bcl-2 and Bax proteins. N-(4-Methoxyphenyl)retinamide, the major 4HPR metabolite, and several other retinoids that bind to nuclear retinoic acid receptors or retinoid X receptors failed to enhance the generation of reactive oxygen species and to induce apoptosis. 4HPR was much less effective in generating reactive oxygen species and in inducing apoptosis in normal human cervical epithelial cells than in C33A cervical carcinoma cells. CONCLUSIONS: Enhancement of the generation of reactive oxygen species may be involved in apoptotic pathway induction by 4HPR.
Asunto(s)
Fenretinida/farmacología , Radicales Libres , Oxígeno/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/fisiopatología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cuello del Útero/citología , Fragmentación del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Epitelio/efectos de los fármacos , Femenino , Fenretinida/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Pirrolidinas/farmacología , Retinoides/farmacología , Tiocarbamatos/farmacología , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/química , Proteína X Asociada a bcl-2RESUMEN
N-(4-Hydroxyphenyl)retinamide (4-HPR, Fenretinide) is a retinoid derivative with antineoplastic activity in various tumor types including prostate carcinoma. The mechanism of action of 4-HPR toxicity is unknown. 4-HPR induces apoptosis in leukemia- and lymphoma-derived cells, neuroblastoma, and small cell lung cancers. The present study was designed to investigate: (a) the mechanism of 4-HPR cytotoxicity in prostate cancer cells; and (b) correlate increased expression of transforming growth factor beta 1 (TGF beta 1) with induction of apoptosis. 4-HPR exposure to PC-3 cells in vitro was associated with apoptosis as evidenced by increased incidence of hypodiploid nuclei in propidium iodide fluorescence histograms and DNA fragmentation. An increase in the percentage of nuclei in the G1 phase of the cell cycle preceded induction of apoptosis. TGF beta 1-increased expression was noted in mRNA levels and in secretion of active TGF beta 1 into culture media. TGF beta 1 and TGF-beta receptor type II detected immunohistochemically were increased in 4-HPR-treated PC-3 cells. Furthermore, 4-HPR-induced cytotoxicity in PC-3 cells was abrogated by the addition of anti-TGF beta 1 antibody. In BT-20 cells, a 4-HPR-resistant breast carcinoma cell line, apoptosis was not observed after exposure to 4-HPR nor was TGF beta 1 expression enhanced in stained cells or in conditioned media. It is concluded that 4-HPR induces the expression of TGF beta 1 in association with the induction of apoptosis.