RESUMEN
Triple-negative breast cancers (TNBCs) are aggressive forms of breast carcinoma associated with a high rate of recidivism. In this paper, we report the production of mammospheres from three lines of TNBC cells and demonstrate that both parthenolide (PN) and its soluble analog dimethylaminoparthenolide (DMAPT) suppressed this production and induced cytotoxic effects in breast cancer stem-like cells, derived from dissociation of mammospheres. In particular, the drugs exerted a remarkable inhibitory effect on viability of stem-like cells. Such an effect was suppressed by N-acetylcysteine, suggesting a role of reactive oxygen species (ROS) generation in the cytotoxic effect. Instead z-VAD, a general inhibitor of caspase activity, was ineffective. Analysis of ROS generation, performed using fluorescent probes, showed that both the drugs stimulated in the first hours of treatment a very high production of hydrogen peroxide. This event was, at least in part, a consequence of activation of NADPH oxidases (NOXs), as it was reduced by apocynin and diphenylene iodinium, two inhibitors of NOXs. Moreover, both the drugs caused downregulation of Nrf2 (nuclear factor erythroid 2-related factor 2), which is a critical regulator of the intracellular antioxidant response. Prolonging the treatment with PN or DMAPT we observed between 12 and 24 h that the levels of both superoxide anion and hROS increased in concomitance with the downregulation of manganese superoxide dismutase and catalase. In addition, during this phase dissipation of mitochondrial membrane potential occurred together with necrosis of stem-like cells. Finally, our results suggested that the effect on ROS generation found in the first hours of treatment was, in part, responsible for the cytotoxic events observed in the successive phase. In conclusion, PN and DMAPT markedly inhibited viability of stem-like cells derived from three lines of TNBCs by inducing ROS generation, mitochondrial dysfunction and cell necrosis.
Asunto(s)
Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Sesquiterpenos/toxicidad , Acetofenonas/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Oligopéptidos/farmacología , Compuestos Onio/farmacología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Triple-negative breast cancers (TNBCs) are clinically aggressive forms associated with a poor prognosis. We evaluated the cytotoxic effect exerted on triple-negative MDA-MB231 breast cancer cells both by parthenolide and its soluble analogue dimethylamino parthenolide (DMAPT) and explored the underlying molecular mechanism. The drugs induced a dose- and time-dependent decrement in cell viability, which was not prevented by the caspase inhibitor z-VAD-fmk. In particular in the first hours of treatment (1-3 h), parthenolide and DMAPT strongly stimulated reactive oxygen species (ROS) generation. The drugs induced production of superoxide anion by activating NADPH oxidase. ROS generation caused depletion of thiol groups and glutathione, activation of c-Jun N-terminal kinase (JNK) and downregulation of nuclear factor kB (NF-kB). During this first phase, parthenolide and DMAPT also stimulated autophagic process, as suggested by the enhanced expression of beclin-1, the conversion of microtubule-associated protein light chain 3-I (LC3-I) to LC3-II and the increase in the number of cells positive to monodansylcadaverine. Finally, the drugs increased RIP-1 expression. This effect was accompanied by a decrement of pro-caspase 8, while its cleaved form was not detected and the expression of c-FLIPS markedly increased. Prolonging the treatment (5-20 h) ROS generation favoured dissipation of mitochondrial membrane potential and the appearance of necrotic events, as suggested by the increased number of cells positive to propidium iodide staining. The administration of DMAPT in nude mice bearing xenografts of MDA-MB231 cells resulted in a significant inhibition of tumour growth, an increment of animal survival and a marked reduction of the lung area invaded by metastasis. Immunohistochemistry data revealed that treatment with DMAPT reduced the levels of NF-kB, metalloproteinase-2 and -9 and vascular endothelial growth factor, while induced upregulation of phosphorylated JNK. Taken together, our data suggest a possible use of parthenolide for the treatment of TNBCs.
Asunto(s)
Autofagia/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Sesquiterpenos/farmacología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Femenino , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , FN-kappa B/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Sesquiterpenos/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The sesquiterpene lactone parthenolide (PN) has recently attracted considerable attention because of its anti-microbial, anti-inflammatory and anticancer effects. However, the mechanism of its cytotoxic action on tumor cells remains scarcely defined. We recently provided evidence that the effect exerted by PN in MDA-MB-231 breast cancer cells was mediated by the production of reactive oxygen species (ROS). The present study shows that PN promoted the phosphorylation of EGF receptor (phospho-EGFR) at Tyr1173, an event which was observed already at 1 h of incubation with 25 µM PN and reached a peak at 8-16 h. This effect seemed to be a consequence of ROS production, because N-acetylcysteine (NAC), a powerful ROS scavenger, prevented the increment of phospho-EGFR levels. In addition fluorescence analyses performed using dihydroethidium demonstrated that PN stimulated the production of superoxide anion already at 2-3 h of incubation and the effect further increased prolonging the time of treatment, reaching a peak at 8-16 h. Superoxide anion production was markedly hampered by apocynin, a well known NADPH oxidase (NOX) inhibitor, suggesting that the effect was dependent on NOX activity. The finding that AG1478, an EGFR kinase inhibitor, substantially blocked both EGFR phosphorylation and superoxide anion production strongly suggested that phosphorylation of EGFR can be responsible for the activation of NOX with the consequent production of superoxide anion. Therefore, EGFR phosphorylation can exert a key role in the production of superoxide anion and ROS induced by PN in MDA-MB-231 cells.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Receptores ErbB/metabolismo , Sesquiterpenos/farmacología , Superóxidos/metabolismo , Acetofenonas/farmacología , Acetilcisteína/metabolismo , Antioxidantes/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Femenino , Humanos , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , FN-kappa B/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Quinazolinas/farmacología , Tirfostinos/farmacologíaRESUMEN
SAHA, an inhibitor of histone deacetylase activity, has been shown to sensitize tumor cells to apoptosis induced by TRAIL, a member of TNF-family. In this paper we investigated the effect of SAHA/TRAIL combination in two breast cancer cell lines, the ERα-positive MCF-7 and the ERα-negative MDA-MB231. Treatment of MDA-MB231 and MCF-7 cells with SAHA in combination with TRAIL caused detachment of cells followed by anoikis, a form of apoptosis which occurs after cell detachment, while treatment with SAHA or TRAIL alone did not produce these effects. The effects were more evident in MDA-MB231 cells, which were chosen for ascertaining the mechanism of SAHA/TRAIL action. Our results show that SAHA decreased the level of c-FLIP, thus favouring the interaction of TRAIL with the specific death receptors DR4 and DR5 and the consequent activation of caspase-8. These effects increased when the cells were treated with SAHA/TRAIL combination. Because z-IEDT-fmk, an inhibitor of caspase-8, prevented both the cleavage of the focal adhesion-kinase FAK and cell detachment, we suggest that activation of caspase-8 can be responsible for both the decrement of FAK and the consequent cell detachment. In addition, treatment with SAHA/TRAIL combination caused dissipation of ΔΨ(m), activation of caspase-3 and decrement of both phospho-EGFR and phospho-ERK1/2, a kinase which is involved in the phosphorylation of BimEL. Therefore, co-treatment also induced decrement of phospho-BimEL and a concomitant increase in the dephosphorylated form of BimEL, which plays an important role in the induction of anoikis. Our findings suggest the potential application of SAHA in combination with TRAIL in clinical trials for breast cancer.
Asunto(s)
Anoicis/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Anoicis/genética , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasas/genética , Caspasas/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , VorinostatRESUMEN
This report shows that histone deacetylase inhibitors (HDACIs) induced apoptosis in human hepatoma HepG2 cells in a dose- and time-dependent manner. Trichostatin A (TSA), ITF2357 and suberoylanilide hydroxamic acid (SAHA), which were very effective agents, caused apoptotic effects after a lag phase of 12-16 h. In order to elucidate the mechanism of HDACIs action in HepG2 cells we have studied the effects of TSA, ITF2357 and SAHA on acetylation of p53 and histones H2A, H2B, H3 and H4. It was observed that HDACIs rapidly induced acetylation of these proteins, being the effects clearly visible already at 30 min of treatment at the same doses which caused apoptosis. Analysis of the immunocomplexes, obtained from nuclear extracts using an antibody against p53, revealed the presence of acetylated p53 together with acetylated forms of histones and histone acetyltransferases p300 and PCAF. Experiments performed using pifithrin-alpha, a reversible inhibitor of p53, showed a correlation between acetylation of p53 and induction of apoptosis. In addition treatment with siRNA against p53 indicated that p53 is involved in the acetylation of histones. In conclusion, this report suggests that complexes constituted by acetylated p53, acetylated histones and coactivators can play a central role in HDACI-induced apoptosis in HepG2 cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Histonas/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Benzotiazoles/farmacología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Daño del ADN , Humanos , Ácidos Hidroxámicos/farmacología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Interferente Pequeño/farmacología , Tolueno/análogos & derivados , Tolueno/farmacología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , VorinostatRESUMEN
Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. This paper shows that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) stimulated at 5-10 microM apoptosis in human hepatoma HepG2 and Huh6 cells, but was ineffective in primary human hepatocytes (PHH). In HepG2 cells SAHA induced the extrinsic apoptotic pathway, increasing the expression of both FasL and FasL receptor and causing the activation of caspase-8. Moreover, SAHA enhanced the level of Bim proteins, stimulated alternative splicing of the Bcl-X transcript with the expression of the proapoptotic Bcl-Xs isoform, induced degradation of Bid into the apoptotic factor t-Bid and dephosphorylation and inactivation of the anti-apoptotic factor Akt. Consequently, SAHA caused loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, activation of caspase-3 and degradation of PARP. Interestingly, a combination of suboptimal doses of SAHA (1 microM) and bortezomib (5-10 nM), a potent inhibitor of 26S proteasome, synergistically induced apoptosis in both HepG2 and Huh6 cells, but was ineffective in PHH. Combined treatment increased with synergistic effects the expression levels of c-Jun, phospho-c-Jun and FasL and the production of Bcl-Xs. These effects were accompanied by activation of Bid, caspase-8 and 3. In conclusion, SAHA stimulated apoptosis in hepatoma cells and exerted a synergistic apoptotic effect when combined with bortezomib. In contrast, these treatments were quite ineffective in inducing apoptosis in PHH. Thus, our results suggest the potential application of the SAHA/bortezomib combination in clinical trials for liver cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Borónicos/metabolismo , Carcinoma Hepatocelular/metabolismo , Ácidos Hidroxámicos/metabolismo , Ácidos Hidroxámicos/farmacología , Pirazinas/metabolismo , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Ácidos Borónicos/farmacología , Bortezomib , Carcinoma Hepatocelular/patología , Caspasa 8/metabolismo , Línea Celular Tumoral/citología , Línea Celular Tumoral/metabolismo , Proteína Ligando Fas/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasoma , Pirazinas/farmacología , Vorinostat , Proteína X Asociada a bcl-2/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The proteasome inhibitor bortezomib is an efficacious apoptotic agent in many tumor cells. This paper shows that bortezomib induced apoptosis in human hepatoma HepG2 cells associated with many modifications in the expression of survival or death factors. Although bortezomib increased the level of the protective factors HSP70 and HSP27, the effects of the drug that favour cell death were predominant. These events include accumulation of c-Jun, phospho-c-Jun and p53; increase in FasL level with activation of caspase-8; changes related to members of Bcl-2 family with increase in the level of pro-apoptotic members and decrease in that of anti-apoptotic ones; dissipation of mitochondrial potential with cytochrome c release and activation of caspase-3. In contrast, Chang liver cells exhibited a very low susceptibility to bortezomib-induced apoptosis, which was accompanied by modest modifications in the expression of apoptotic factors. In HepG2 cells bortezomib markedly increased AP-1 activity and the expression of its transcriptional targets such as c-Jun, FasL, BimEL, which are involved in apoptosis. Moreover, AP-1 induced its own production by increasing c-Jun content in the composition of the same AP-1 complex. In addition, bortezomib caused activation of JNK1, which in turn increased the level of phospho-c-Jun as well as stimulated the activation of caspase-3 and t-Bid, two fundamental apoptotic factors. Interestingly, siRNA silencing of c-Jun or JNK1 reduced HepG2 cell susceptibility to apoptosis and prevented the increase in AP-1 activity. Both JNK-1 and AP-1 thus exerted a crucial role in bortezomib-induced apoptosis. Differently, in Chang liver cells the different composition of AP-1 complex as well as the failure of JNK activation seemed to be responsible for the low susceptibility to apoptosis. Given the high susceptibility of hepatoma cells to bortezomib, our results suggest the potential application of this compound in clinical trials for liver cancers.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Ácidos Borónicos/farmacología , Neoplasias Hepáticas/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Inhibidores de Proteasas/farmacología , Pirazinas/farmacología , Factor de Transcripción AP-1/metabolismo , Bortezomib , Caspasa 8 , Caspasas/metabolismo , Línea Celular , Línea Celular Tumoral , Proteína Ligando Fas , Proteínas de Choque Térmico/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transducción de Señal , Factores de Necrosis Tumoral/metabolismoRESUMEN
The transcriptional coactivator CBP displays an intrinsic histone acetyl transferase (HAT) activity which seems to participate in transcriptional activation through the destabilization of nucleosome structure. CBP is involved in the activity of several transcription factors that are nuclear endpoints of intracellular signal transduction pathways. In some instances, the transcription factors are phosphorylated upon cell activation, which induces their interaction with CBP. CBP itself is a phosphoprotein and can be phosphorylated by cycle-dependent kinases or by MAP kinases. Here we show that CBP phosphorylation by p44 MAP kinase/ERK1 results in the stimulation of its HAT enzymatic activity. The p44 MAP kinase/ERK1 phosphorylation sites are located in the C-terminal part of the protein, outside of the HAT domain. These sites are required for enzymatic stimulation, suggesting that phosphorylation by p44 MAP kinase/ERK1 induces a conformational change of the CBP molecule. Our data suggest that, in some instances, CBP itself might be a target for signal transduction pathways.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Células 3T3 , Acetiltransferasas/química , Adenosina Trifosfato/metabolismo , Animales , Proteína de Unión a CREB , Activación Enzimática , Histona Acetiltransferasas , Histonas/metabolismo , Ratones , Proteína Quinasa 3 Activada por Mitógenos , Modelos Genéticos , Proteínas Nucleares/química , Nucleosomas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Transducción de Señal , Factores de Tiempo , Transactivadores/químicaRESUMEN
Addition of nerve growth factor (NGF) to PC12 cells promotes neuronal differentiation while inhibiting cell proliferation. In order to understand how NGF exerts its antimitogenic effect during differentiation, we have studied the mechanism by which this factor activates the promoter of the CDK inhibitor p21W4F1/CIP1. The minimal region of the p21 promoter required for the NGF-induction was mapped to a contiguous stretch of 10 bp located 83 bases upstream of the transcription initiation site. This GC-rich region was shown to interact specifically with the transcription factor Sp1 and the related protein Sp3, in either exponentially-growing or NGF-treated PC12 cells. The addition of NGF resulted in an accumulation of the transcriptional co-activator p300 in complexes associated with the NGF-responsive region. Transcriptional activity of Sp1, Sp3 and p300 was specifically induced by NGF in a Gal4-fusion assay, indicating that induction of p21 during neuronal differentiation may involve regulation of the activity of these factors by NGF. Furthermore, p300 was able to act as a co-activator for Sp1-mediated transcriptional activation in PC12 cells, suggesting that p300 and Sp1 may cooperate in activating p21 transcription during the withdrawal of neuronal precursors from the cell cycle. This hypothesis is supported by experiments showing that p300 and Sp1 form complexes in PC12 cells.