RESUMEN
PURPOSE: To investigate the effect of TAP7f, a penicillin derivative previously characterized as a potent antitumor agent that promotes ER stress and apoptosis, in combination with thapsigargin, an ER stress inducer, on melanoma cells. METHODS: The synergistic antiproliferative effect of TAP7f in combination with thapsigargin was studied in vitro in murine B16-F0 melanoma cells, and in human A375 and SB2 melanoma cells. In vivo assays were performed with C57BL/6J mice challenged with B16-F0 cells. Immunofluorescence and Western blot assays were carried out to characterize the induction of ER stress and apoptosis. Necrotic tumor areas and the potential toxicity of the combined therapy were examined by histological analysis of tissue sections after hematoxylin-eosin staining. RESULTS: In vitro, the combination of TAP7f with thapsigargin synergistically inhibited the proliferation of murine B16-F0, and human A375 and SB2 melanoma cells. When non-inhibitory doses of each drug were simultaneously administered to C57BL/6J mice challenged with B16-F0 cells, a 50% reduction in tumor volumes was obtained in the combined group. An apoptotic response characterized by higher expression levels of Baxenhanced PARP-1 cleavage and the presence of active caspase 3 was observed in tumors from the combined treatment. In addition, higher expression levels of GADD153/CHOP and ATF4 were found in tumors of mice treated with both drugs with respect to each drug used alone, indicating the induction of an ER stress response. No signs of tissue toxicity were observed in histological sections of different organs extracted from mice receiving the combination. CONCLUSION: The synergistic and effective antitumor action of TAP7f in combination with thapsigargin could be considered as a potential therapeutic strategy for melanoma treatment.
Asunto(s)
Antineoplásicos , Melanoma , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Apoptosis , Caspasa 3/metabolismo , Línea Celular Tumoral , Melanoma/patología , Ratones Endogámicos C57BL , Penicilinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Tapsigargina/farmacologíaRESUMEN
Nowadays, the nutraceutical agent sulforaphane (SFN) shows great versatility in turning on different cellular responses. Mainly, this isothiocyanate acts as a master regulator of cellular homeostasis due to its antioxidant response and cytoplasmic, mitochondrial, and endoplasmic reticulum (ER) protein modulation. Even more, SFN acts as an effective strategy to counteract oxidative stress, apoptosis, and ER stress, among others as seen in different injury models. Particularly, ER stress is buffered by the unfolded protein response (UPR) activation, which is the first instance in orchestrating the recovery of ER function. Interestingly, different studies highlight a close interrelationship between ER stress and oxidative stress, two events driven by the accumulation of reactive oxygen species (ROS). This response inevitably perpetuates itself and acts as a vicious cycle that triggers the development of different pathologies, such as cardiovascular diseases, neurodegenerative diseases, and others. Accordingly, it is vital to target ER stress and oxidative stress to increase the effectiveness of clinical therapies used to treat these diseases. Therefore, our study is focused on the role of SFN in preserving cellular homeostasis balance by regulating the ER stress response through the Nrf2-modulated antioxidant pathway.
Asunto(s)
Antioxidantes , Isotiocianatos , Antioxidantes/metabolismo , Antioxidantes/farmacología , Apoptosis , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Homeostasis , Isotiocianatos/metabolismo , Isotiocianatos/farmacología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Sulfóxidos , Respuesta de Proteína DesplegadaRESUMEN
The Trypanosomatidae family encompasses many unicellular organisms responsible of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by Trypanosoma brucei brucei (T. brucei), Trypanosoma equiperdum (T. equiperdum) and Trypanosoma evansi (T. evansi), have a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world. Similarly, to all organisms, the maintenance of Ca2+ homeostasis is vital for these parasites, and the mechanism involved in the intracellular Ca2+ regulation have been widely described. However, the evidences related to the mechanisms responsible for the Ca2+ entry are scarce. Even more, to date the presence of a store-operated Ca2+ channel (SOC) has not been reported. Despite the apparent absence of Orai and STIM-like proteins in these parasites, in the present work we demonstrate the presence of a store-operated Ca2+-entry (SOCE) in T. equiperdum, using physiological techniques. This Ca2+-entry is induced by thapsigargin (TG) and 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), and inhibited by 2-aminoethoxydiphenyl borate (2APB). Additionally, the use of bioinformatics techniques allowed us to identify putative transient receptor potential (TRP) channels, present in members of the Trypanozoon family, which would be possible candidates responsible for the SOCE described in the present work in T. equiperdum.
Asunto(s)
Calcio/metabolismo , Proteínas Sensoras del Calcio Intracelular/metabolismo , Proteínas Protozoarias/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Trypanosoma/metabolismo , Animales , Compuestos de Boro/farmacología , Quelantes del Calcio/química , Biología Computacional/métodos , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/química , Fura-2/química , Expresión Génica , Homeostasis/genética , Hidroquinonas/farmacología , Proteínas Sensoras del Calcio Intracelular/genética , Manganeso/metabolismo , Proteínas Protozoarias/genética , Tapsigargina/farmacología , Canales de Potencial de Receptor Transitorio/genética , Trypanosoma/efectos de los fármacos , Trypanosoma/genética , Tripanosomiasis/parasitologíaRESUMEN
The Cerebral Dopamine Neurotrophic Factor (CDNF) is a neurotrophic factor that has a protective effect in cell and animal models of several neurodegenerative diseases. The molecular mechanism of the protective effect of CDNF is unclear. Many neurodegenerative diseases have been related to a proteostasis dysregulation in the endoplasmic reticulum (ER). A failure of proteostasis produces ER stress, triggering the unfolded protein response (UPR) and, in the long-term, induces cell death. An adaptive UPR solves ER stress by attenuating protein synthesis, inducing chaperones expression, and degradation of misfolded proteins. Since CDNF is an ER resident protein, we investigated whether the role of CDNF is to regulate ER proteostasis. To this end, we determined the effect of CDNF in thapsigargin-induced ER stress in HEK293-T cells and cultured hippocampal neurons. Our results show that CDNF improved the viability of HEK293-T cells exposed to thapsigargin. CDNF increased levels of protective proteins of the early UPR, such as BiP, ATF4, ATF6, and XBP-1 in both HEK293-T cells and neurons. Conversely, expression of CDNF attenuated ER stress-induced apoptotic proteins, CHOP and cleaved caspase-3 in HEK293-T cells and neurons. A mutant CDNF lacking the ER retention sequence failed to protect against ER stress. In conclusion, CDNF regulates proteostasis in the ER by inducing the adaptive UPR response and inhibiting apoptotic pathways triggered by ER stress. We propose that neuroprotection induced by CDNF is mediated by regulating ER proteostasis.
Asunto(s)
Muerte Celular , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Respuesta de Proteína Desplegada , Animales , Apoptosis/efectos de los fármacos , Biología Computacional/métodos , Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células HEK293 , Humanos , Factores de Crecimiento Nervioso/farmacología , Células Piramidales/metabolismo , RatasRESUMEN
It has been shown that 2-APB is a nonspecific modulator of ion channel activity, while most of the channels are inhibited by this compound, there are few examples of channels that are activated by 2-APB. Additionally, it has been shown that, 2-APB leads to a reduction in the luminal endoplasmic reticulum Ca2+ level ([Ca2+]ER) and we have carried out simultaneous recordings of both [Ca2+]i and the [Ca2+]ER in HeLa cell suspensions to assess the mechanism involved in this effect. This approach allowed us to determine that 2-APB induces a reduction in the [Ca2+]ER by activating an ER-resident Ca2+ permeable channel more than by inhibiting the activity of SERCA pumps. Interestingly, this effect of 2-APB of reducing the [Ca2+]ER is auto-limited because depends on a replete ER Ca2+ store; a condition that thapsigargin does not require to decrease the [Ca2+]ER. Additionally, our data indicate that the ER Ca2+ permeable channel activated by 2-APB does not seem to participate in the ER Ca2+ leak revealed by inhibiting SERCA pump with thapsigargin. This work suggests that, prolonged incubations with even low concentrations of 2-APB (5µM) would lead to the reduction in the [Ca2+]ER that might explain the inhibitory effect of this compound on those signals that require Ca2+ release from the ER store.
Asunto(s)
Compuestos de Boro/farmacología , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Células HeLa , Humanos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tapsigargina/farmacologíaRESUMEN
In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca(2+)-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 µM TG or 25 µM 2',5'-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca(2+) and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG.
Asunto(s)
ATPasas Transportadoras de Calcio/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Bombas Iónicas/efectos de los fármacos , Tapsigargina/farmacología , Trypanosoma/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/inmunología , Retículo Endoplásmico/enzimología , Enfermedades de los Caballos/parasitología , Caballos , Bombas Iónicas/metabolismo , Masculino , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Trypanosoma/efectos de los fármacos , Trypanosoma/fisiología , Tripanosomiasis/parasitología , Tripanosomiasis/veterinariaRESUMEN
Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.
Asunto(s)
ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Entamoeba/efectos de los fármacos , Entamoeba/crecimiento & desarrollo , Proteínas Protozoarias/antagonistas & inhibidores , Esporas Protozoarias/efectos de los fármacos , Esporas Protozoarias/crecimiento & desarrollo , ATPasas Transportadoras de Calcio/análisis , ATPasas Transportadoras de Calcio/genética , Calreticulina/análisis , Inhibidores Enzimáticos/metabolismo , Indoles/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genética , Tapsigargina/metabolismo , Vesículas Transportadoras/químicaRESUMEN
Ryanodine is a cell permeant plant alkaloid that binds selectively and with high affinity to ryanodine receptor (RyR) Ca(2+) release channels. Sub-micromolar ryanodine concentrations activate RyR channels while micromolar concentrations are inhibitory. Several reports indicate that neuronal synaptic plasticity, learning and memory require RyR-mediated Ca(2+)-release, which is essential for muscle contraction. The use of micromolar (inhibitory) ryanodine represents a common strategy to suppress RyR activity in neuronal cells: however, micromolar ryanodine promotes RyR-mediated Ca(2+) release and endoplasmic reticulum Ca(2+) depletion in muscle cells. Information is lacking in this regard in neuronal cells; hence, we examined here if addition of inhibitory ryanodine elicited Ca(2+) release in primary hippocampal neurons, and if prolonged incubation of primary hippocampal cultures with inhibitory ryanodine affected neuronal ER calcium content. Our results indicate that inhibitory ryanodine does not cause Ca(2+) release from the ER in primary hippocampal neurons, even though ryanodine diffusion should produce initially low intracellular concentrations, within the RyR activation range. Moreover, neurons treated for 1 h with inhibitory ryanodine had comparable Ca(2+) levels as control neurons. These combined findings imply that prolonged incubation with inhibitory ryanodine, which effectively abolishes RyR-mediated Ca(2+) release, preserves ER Ca(2+) levels and thus constitutes a sound strategy to suppress neuronal RyR function.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Hipocampo/citología , Neuronas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Rianodina/farmacología , Animales , Ionóforos de Calcio/farmacología , Células Cultivadas , Cresoles/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ionomicina/farmacología , Neuronas/efectos de los fármacos , Ratas Sprague-Dawley , Rianodina/agonistas , Tapsigargina/farmacologíaRESUMEN
Cardiac fibroblast (CF) survival is important for the maintenance of the extracellular matrix homeostasis in the heart; providing a functional support to cardiomyocytes necessary for the correct myocardial function. Endoplasmic reticulum (ER) stress causes cellular dysfunction and cell death by apoptosis; and thapsigargin is a well-known ER stress inducer. On the other hand, the chemical chaperone, 4-phenylbutyric acid (4-PBA) had showed to prevent ER stress; however, in cardiac fibroblast both the ER stress induced by thapsigargin and prevention by 4-PBA, have not been studied in detail. Neonate rat CF were treated with thapsigargin in presence or absence of 4-PBA, and cell viability was evaluated by trypan blue exclusion and apoptosis by flow cytometry; whereas CHOP, BIP, PDI, ATF4 and procollagen protein levels were assessed by western blot. In CF, thapsigargin triggered the unfolded protein response detected by early increases in ATF4, CHOP, PDI and BIP protein levels as well as, the accumulation of intracellular procollagen. Thapsigargin also stimulated CF death in a time and concentration-dependent manner. ER stress, CF death and apoptosis induced by thapsigargin were prevented by 4-PBA. Conclusion our data suggest that 4-PBA prevent ER stress, intracellular procollagen accumulation, CF death and apoptosis induced by thapsigargin.
Asunto(s)
Fenilbutiratos/farmacología , Tapsigargina/toxicidad , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Miocitos Cardíacos/citología , Procolágeno/metabolismo , Ratas , Ratas Sprague-Dawley , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.