RESUMO
The leucine-rich repeat containing 8A (LRRC8A) protein is an essential component of the volume-sensitive organic anion channel (VSOAC), and using pharmacological anion channel inhibitors (NS3728, DIDS) and LRRC8A siRNA we have investigated its role in development of Cisplatin resistance in human ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of p21(Waf1/Cip1), Bax, Noxa, MDM2, and activation of Caspase-9/-3. In contrast, Cisplatin-resistant cells do not enter apoptosis, i.e., their p53 and downstream signaling are reduced and caspase activity unaltered following Cisplatin exposure. Reduced LRRC8A expression and VSOAC activity are previously shown to correlate with Cisplatin resistance, and here we demonstrate that pharmacological inhibition and transient knockdown of LRRC8A reduce the protein level of p53, MDM2, and p21(Waf1/Cip1) as well as Caspase-9/-3 activation in Cisplatin-sensitive cells. Cisplatin resistance is accompanied by reduction in total LRRC8A expression (A2780) or LRRC8A expression in the plasma membrane (A549). Activation of Caspase-3 dependent apoptosis by TNFα-exposure or hyperosmotic cell shrinkage is almost unaffected by pharmacological anion channel inhibition. Our data indicate 1) that expression/activity of LRRC8A is essential for Cisplatin-induced increase in p53 protein level and its downstream signaling, i.e., Caspase-9/-3 activation, expression of p21(Waf1/Cip1) and MDM2; and 2) that downregulation of LRRC8A-dependent osmolyte transporters contributes to acquirement of Cisplatin resistance in ovarian and lung carcinoma cells. Activation of LRRC8A-containing channels is upstream to apoptotic volume decrease as hypertonic cell shrinkage induces apoptosis independent of the presence of LRRC8A.
Assuntos
Adenocarcinoma Bronquioloalveolar/tratamento farmacológico , Antineoplásicos/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Cisplatino/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células A549 , Adenocarcinoma Bronquioloalveolar/enzimologia , Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma Bronquioloalveolar/patologia , Proteínas de Transporte de Ânions/antagonistas & inibidores , Proteínas de Transporte de Ânions/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 9/genética , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/metabolismo , Tamanho Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Moduladores de Transporte de Membrana/farmacologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
OBJECTIVES: Novel treatments for pancreatic ductal adenocarcinoma (PDAC) are severely needed. The aim of this work was to explore the roles of H-lactate monocarboxylate transporters 1 and 4 (MCT1 and MCT4) in PDAC cell migration and invasiveness. METHODS: Monocarboxylate transporter expression, localization, activity, and function were explored in human PDAC cells (MIAPaCa-2, Panc-1, BxPC-3, AsPC-1) and normal human pancreatic ductal epithelial (HPDE) cells, by quantitative polymerase chain reaction, immunoblotting, immunocytochemistry, lactate flux, migration, and invasion assays. RESULTS: MCT1 and MCT4 (messenger RNA, protein) were robustly expressed in all PDAC lines, localizing to the plasma membrane. Lactate influx capacity was highest in AsPC-1 cells and lowest in HPDE cells and was inhibited by the MCT inhibitor α-cyano-4-hydroxycinnamate (4-CIN), MCT1/MCT2 inhibitor AR-C155858, or knockdown of MCT1 or MCT4. PDAC cell migration was largely unaffected by MCT1/MCT2 inhibition or MCT1 knockdown but was reduced by 4-CIN and by MCT4 knockdown (BxPC-3). Invasion measured in Boyden chamber (BxPC-3, Panc-1) and spheroid outgrowth (BxPC-3) assays was attenuated by 4-CIN and AR-C155858 and by MCT1 or MCT4 knockdown. CONCLUSIONS: Human PDAC cells exhibit robust MCT1 and MCT4 expression and partially MCT1- and MCT4-dependent lactate flux. PDAC cell migration is partially dependent on MCT4; and invasion, on MCT1 and MCT4. Inhibition of MCT1 and MCT4 may have clinical relevance in PDAC.
Assuntos
Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/genética , Simportadores/genética , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Microscopia de Fluorescência , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores/metabolismoRESUMO
The aim of this project was to analyze the regulation of p53 expression in NIH3T3 fibroblasts under the influence of increasing hyperosmotic stress. Expression of p53 showed a biphasic response pattern in NIH3T3 cells under increasing osmotic stress (337 mOsm to 737 mOsm) with a maximum at 587 mOsm. Under isotonic conditions p53 expression increased after addition of the proteasome inhibitor MG132 indicating that cellular p53 levels in unperturbed cells is kept low by proteasomal degradation. However, under hypertonic conditions p53 synthesis as well as p53 degradation were significantly reduced and it is demonstrated that the increase in p53 expression observed when tonicity is increased from 337 to 587 mOsm reflects that degradation is more inhibited than synthesis, whereas the decrease in p53 expression at higher tonicities reflects that synthesis is more inhibited than degradation. The activity of the p53 regulating proteins p38 MAP kinase and the ubiquitin ligase MDM2 were studied as a function of increasing osmolarity. MDM2 protein expression was unchanged at all osmolarities, whereas MDM2 phosphorylation (Ser(166)) increased at osmolarities up to 537 mOsm and remained constant at higher osmolarities. Phosphorylation of p38 increased at osmolarities up to 687 mOsm which correlated with an increased phosphorylation of p53 (Ser(15)) and the decreased p53 degradation. Caspase-3 activity increased gradually with hypertonicity and at 737 mOsm both Caspase-3 activity and annexin V binding are high even though p53 expression and activity are low, indicating that initiation of apoptosis under severe hypertonic conditions is not strictly controlled by p53.
RESUMO
BACKGROUND/AIMS: Altered expression of the integrin family of cell adhesion receptors has been associated with initiation, progression, and metastasis of solid tumors as well as in the development of chemoresistance. Here, we investigated the role of integrins, in particular integrin ß1, in cell volume regulation and drug-induced apoptosis in adherent and non-adherent Ehrlich ascites cell lines. METHODS: Adhesion phenotypes were verified by colorimetric cell-adhesion-assay. Quantitative real-time PCR and western blot were used to compare expression levels of integrin subunits. Small interfering RNA was used to silence integrin ß1 expression. Regulatory volume decrease (RVD) after cell swelling was studied with calcein-fluorescence-self-quenching and Coulter counter analysis. Taurine efflux was estimated with tracer technique. Caspase assay was used to determine apoptosis. RESULTS: We show that adherent cells have stronger fibronectin binding and a significantly increased expression of integrin α5, αv, and ß1 at mRNA and protein level, compared to non-adherent cells. Knockdown of integrin ß1 reduced RVD of the adherent but not of the non-adherent cells. Efflux of taurine was unaffected. In contrast to non-adherent, adherent cells exhibited chemoresistance to chemotherapeutic drugs (cisplatin and gemcitabine). However, knockdown of integrin ß1 promoted cisplatin-induced caspase activity in adherent cells. CONCLUSION: Our data identifies integrin ß1 as a part of the osmosensing machinery and regulator of cisplatin resistance in adherent Ehrlich cells.
Assuntos
Carcinoma de Ehrlich/metabolismo , Resistencia a Medicamentos Antineoplásicos , Integrina beta1/genética , Integrina beta1/metabolismo , Osmose , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma de Ehrlich/genética , Carcinoma de Ehrlich/patologia , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Cisplatino/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Fibronectinas/metabolismo , Camundongos , Taurina/metabolismo , GencitabinaRESUMO
The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10-30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2-10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 µM) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/577 and Tyr861, yet not at Tyr397, and inhibited shrinkage-induced NKCC1 activity by â¼50%. The FAK inhibitor PF-573,228 augmented shrinkage-induced Src phosphorylation, and inhibited shrinkage-induced NKCC1 activity by â¼15%. The apparent role of Src in NKCC1 activation did not reflect phosphorylation of myosin light chain kinase (MLC), which was unaffected by shrinkage and by PP2, but may involve Jak2, a known target of Src, which was rapidly activated by osmotic shrinkage and inhibited by PP2. Collectively, our findings suggest a major role for Src and possibly the Jak2 axis in shrinkage-activation of NKCC1 in NIH3T3 cells, whereas no evidence was found for major roles for FAK and MLC in this process.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Osmose/fisiologia , Fosforilação/fisiologia , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Janus Quinase 2/metabolismo , Camundongos , Quinase de Cadeia Leve de Miosina/metabolismo , Células NIH 3T3 , Vinculina/metabolismoRESUMO
The review describes molecular and functional properties of the volume regulated anion channel and Ca(2+)-dependent Cl(-) channels belonging to the anoctamin family with emphasis on physiological importance of these channels in regulation of cell volume, cell migration, cell proliferation, and programmed cell death. Finally, we discuss the role of Cl(-) channels in various diseases.
Assuntos
Ânions/metabolismo , Cálcio/metabolismo , Fenômenos Fisiológicos Celulares , Canais de Cloreto/metabolismo , Animais , HumanosRESUMO
The importance of the TRPV4 channel for human physiology has been highlighted in recent years with the identification of an increasing number of hereditary diseases associated with mutations of this channel. However, the functional understanding of TRPV4 associated pathologies remains a puzzle due to incomplete understanding of the polymodal regulation of TRPV4 channels and lack of insight into the structure-function relationship of the channel. In this work, we identified a series of highly conserved aromatic residues in transmembrane (TM) helices 5-6 with profound importance for TRPV4 activity. Substituting F617, Y621 or F624 in TM5 with leucine reduced channel sensitivity to the agonist 4α-PDD and heat, yet two of these mutants - F617L and Y621L - showed increased activation in response to cell swelling. In TM6, a Y702L mutation significantly reduced sensitivity to all of the above stimuli. In conclusion, we have identified residues in TM5-6 which differentially affect heat and agonist activation, and we have demonstrated distinct activation pathways for 4α-PDD and osmolarity.
Assuntos
Soluções Hipotônicas/farmacologia , Ésteres de Forbol/farmacologia , Mutação Puntual/genética , Porinas/fisiologia , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/genética , Sequência de Aminoácidos , Cálcio/fisiologia , Carcinógenos/farmacologia , Fenômenos Eletrofisiológicos , Células HEK293 , Humanos , Dados de Sequência Molecular , Concentração Osmolar , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/genética , Canais de Cátion TRPV/químicaRESUMO
BACKGROUND/AIMS: Regulatory volume decrease (RVD) in response to acute cell swelling is well described and KCNK5 (also known as TASK-2 or K2P5.1) has been shown to be the volume sensitive K(+) channel in Ehrlich cells. Very little is, on the other hand, known about the effects of long-term hypotonicity on expression and function of KCNK5, thus we have investigated the effect of long-term hypotonicity (24h - 48h) on KCNK5 in Ehrlich cells on the mRNA, protein and physiological levels. METHODS: Physiological effects of long-term hypotonicity were measured using patch-clamp and Coulter counter techniques. Expression patterns of KCNK5 on mRNA and protein levels were established using real-time qPCR and western blotting respectively. RESULTS: The maximum swelling-activated current through KCNK5 was significantly decreased upon 48h of hypotonicity and likewise the RVD response was significantly impaired after both 24 and 48h of hypotonic stimulation. No significant differences in the KCNK5 mRNA expression patterns between control and stimulated cells were observed, but a significant decrease in the KCNK5 protein level 48h after stimulation was found. CONCLUSION: The data suggest that the strong physiological impairment of KCNK5 in Ehrlich cells after long-term hypotonic stimulation is predominantly due to down-regulation of the KCNK5 protein synthesis.
Assuntos
Carcinoma de Ehrlich/metabolismo , Pressão Osmótica/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Carcinoma de Ehrlich/patologia , Tamanho Celular , Regulação para Baixo , Regulação da Expressão Gênica , Camundongos , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/genética , Células Tumorais CultivadasRESUMO
The present work was initiated to investigate regulation of the taurine transporter TauT by reactive oxygen species (ROS) and the tonicity-responsive enhancer binding protein (TonEBP) in NIH3T3 mouse fibroblasts during acute and long-term (4 h) exposure to low-sodium/hypo-osmotic stress. Taurine influx is reduced following reduction in osmolarity, keeping the extracellular Na(+) concentration constant. TonEBP activity is unaltered, whereas TauT transcription as well as TauT activity are significantly reduced under hypo-osmotic conditions. In contrast, TonEBP activity and TauT transcription are significantly increased following hyperosmotic exposure. Swelling-induced ROS production in NIH3T3 fibroblasts is generated by NOX4 and by increasing total ROS, by either exogenous application of H(2)O(2) or overexpressing NOX4, we demonstrate that TonEBP activity and taurine influx are regulated negatively by ROS under hypo-osmotic, low-sodium conditions, whereas the TauT mRNA level is unaffected. Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics. Thus, swelling-induced ROS production could account for the reduced taurine uptake under low-sodium/hypo-osmotic conditions by direct modulation of TauT.
Assuntos
Fibroblastos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Estresse Fisiológico/fisiologia , Animais , Células Cultivadas , Regulação para Baixo , Peróxido de Hidrogênio/metabolismo , Soluções Hipertônicas , Soluções Hipotônicas , Cinética , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Células NIH 3T3 , Osmose , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Sódio/metabolismo , Taurina/genética , Taurina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Equilíbrio HidroeletrolíticoRESUMO
This mini review outlines studies of cell volume regulation in two closely related mammalian cell lines: nonadherent Ehrlich ascites tumour cells (EATC) and adherent Ehrlich Lettre ascites (ELA) cells. Focus is on the regulatory volume decrease (RVD) that occurs after cell swelling, the volume regulatory ion channels involved, and the mechanisms (cellular signalling pathways) that regulate these channels. Finally, I shall also briefly review current investigations in these two cell lines that focuses on how changes in cell volume can regulate cell functions such as cell migration, proliferation, and programmed cell death.
Assuntos
Canais Iônicos/fisiologia , Animais , Apoptose , Carcinoma de Ehrlich/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Tamanho Celular , Canais de Cloreto/metabolismo , Canais de Cloreto/fisiologia , Canais Iônicos/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/fisiologiaRESUMO
The swelling-activated K(+) currents (I(K,vol)) in Ehrlich ascites tumor cells (EATC) has been reported to be through the two-pore domain (K(2p)), TWIK-related acid-sensitive K(+) channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by I(K,vol) indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel to a lower cell volume. Swelling-activated K(+) efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium, it is demonstrated that mpV(pic) increased the volume-sensitive part of the K(+) efflux 1.3 times. To exclude K(+) efflux via a KCl cotransporter, cellular Cl(-) was substituted with NO(3)(-). Also under these conditions K(+) efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved in the activation of the volume-sensitive K(+) channel, whereas tyrosine phosphatases appears to be involved in inactivation of the channel. Overexpressing TASK-2 in human embryonic kidney (HEK)-293 cells increased the RVD rate and reduced the volume set point. TASK-2 has tyrosine sites, and precipitation of TASK-2 together with Western blotting and antibodies against phosphotyrosines revealed a cell swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors of the epidermal growth factor receptor tyrosine kinases had no effect on RVD, whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the RVD by 40%. It is suggested that the cytokine receptor-coupled JAK/STAT pathway is upstream of the swelling-induced phosphorylation and activation of TASK-2 in EATC.
Assuntos
Tamanho Celular , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Transdução de Sinais/fisiologia , Tirosina/metabolismo , Animais , Linhagem Celular , Inibidores Enzimáticos/metabolismo , Feminino , Genisteína/metabolismo , Humanos , Soluções Hipotônicas , Janus Quinases/metabolismo , Camundongos , Técnicas de Patch-Clamp , Fosforilação , Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Fatores de Transcrição STAT/metabolismoRESUMO
Channels and transporters of monovalent ions are increasingly suggested as putative anticarcinogenic targets. However, the mechanisms involved in modulation of proliferation by monovalent ions are poorly understood. Here, we investigated the role of K+, Na+, and Cl(-) ions for the proliferation of Ehrlich Lettre ascites (ELA) cells. We measured the intracellular concentration of each ion in G(0), G(1), and S phases of the cell cycle following synchronization by serum starvation and release. We show that intracellular concentrations and content of Na+ and Cl(-) were reduced in the G(0)-G(1) phase transition, followed by an increased content of both ions in S phase concomitant with water uptake. The effect of substituting extracellular monovalent ions was investigated by bromodeoxyuridine incorporation and showed marked reduction after Na+ and Cl(-) substitution. In spectrofluorometric measurements with the pH-sensitive dye BCECF, substitution of Na+ was observed to upregulate the activity of the Na+/H+ exchanger NHE1 as well as of Na+-independent acid extrusion mechanisms, facilitating intracellular pH (pH(i)) recovery after acid loading and increasing pH(i). Results using the potential sensitive dye DiBaC4(3) showed a reduced Cl(-) conductance in S compared with G(1) followed by transmembrane potential (E(m)) hyperpolarization in S. Cl(-) substitution by impermeable anions strongly inhibited proliferation and increased free, intracellular Ca2+ ([Ca2+]i), whereas a more permeable anion had little effect. Western blots showed reduced chloride intracellular channel CLIC1 and chloride channel ClC-2 expression in the plasma membrane in S compared with G(1). Our results suggest that Na+ regulates ELA cell proliferation by regulating intracellular pH while Cl(-) may regulate proliferation by fine-tuning of E(m) in S phase and altered Ca2+ signaling.
Assuntos
Carcinoma de Ehrlich/patologia , Cloretos/fisiologia , Potássio/fisiologia , Sódio/fisiologia , Animais , Ânions , Cálcio/metabolismo , Cátions Monovalentes , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/metabolismo , Fase G1 , Concentração de Íons de Hidrogênio , Meglumina/farmacologia , Transporte Proteico , Fase de Repouso do Ciclo Celular , Fase S , Trocadores de Sódio-Hidrogênio/metabolismo , Água/fisiologiaRESUMO
Platinum compounds are used in the treatment of cancer. We demonstrate that cisplatin-induced (10 µM) apoptosis (caspase-3 activity) is pronounced within 18 hours in non-adherent Ehrlich ascites tumour cells (EATC), whereas there is no increase in caspase-3 activity in the adherent Ehrlich Lettré ascites tumour cells (ELA). Loss of KCl and cell shrinkage are hallmarks in apoptosis and has been shown in EATC. However, we find no reduction in cell volume and only a minor loss of K(+) which is accompanied by net uptake of Na(+) following 18 hours cisplatin exposure in ELA. Glutathione and taurine have previously been demonstrated to protect cells from apoptosis. We find, however, that increase or decrease in the cellular content of glutathione and taurine has no effect on cisplatin-induced cell death in EATC and ELA. Nevertheless, knock-down of the taurine transporter TauT leads to a significant increase in apoptosis in ELA following cisplatin exposure. We find that cytosolic accumulation of cisplatin is similar in EATC and ELA. However, the nuclear accumulation and DNA-binding of cisplatin is significant lower in ELA compared to EATC. We suggest three putative reasons for the observed cisplatin insensitivity in the adherent tumor cells (ELA) compared to the non-adherent tumor cells (EATC): less nuclear cisplatin accumulation, increased TauT activity, and decreased anion and water loss.
Assuntos
Antineoplásicos/toxicidade , Apoptose , Cisplatino/toxicidade , Animais , Caspase 3/metabolismo , Tamanho Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Glutationa/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , MicroRNAs/metabolismo , Potássio/metabolismo , Taurina/metabolismo , Células Tumorais CultivadasRESUMO
Addition of H(2)O(2) (0.5 mM) to Ehrlich ascites tumor cells under isotonic conditions results in a substantial (22 +/- 1%) reduction in cell volume within 25 min. The cell shrinkage is paralleled by net loss of K(+), which was significant within 8 min, whereas no concomitant increase in the K(+) or Cl(-) conductances could be observed. The H(2)O(2)-induced cell shrinkage was unaffected by the presence of clofilium and clotrimazole, which blocks volume-sensitive and Ca(2+)-activated K(+) channels, respectively, and is unaffected by a raise in extracellular K(+) concentration to a value that eliminates the electrochemical driving force for K(+). On the other hand, the H(2)O(2)-induced cell shrinkage was impaired in the presence of the KCl cotransport inhibitor (dihydro-indenyl)oxyalkanoic acid (DIOA), following substitution of NO(3)(-) for Cl(-), and when the driving force for KCl cotransport was omitted. It is suggested that H(2)O(2) activates electroneutral KCl cotransport in Ehrlich ascites tumor cells and not K(+) and Cl(-) channels. Addition of H(2)O(2) to hypotonically exposed cells accelerates the regulatory volume decrease and the concomitant net loss of K(+), whereas no additional increase in the K(+) and Cl(-) conductance was observed. The effect of H(2)O(2) on cell volume was blocked by the serine-threonine phosphatase inhibitor calyculin A, indicating an important role of serine-threonine phosphorylation in the H(2)O(2)-mediated activation of KCl cotransport in Ehrlich cells. In contrast, addition of H(2)O(2) to adherent cells, e.g., Ehrlich Lettré ascites cells, a subtype of the Ehrlich ascites tumor cells, and NIH3T3 mouse fibroblasts increased the K(+) and Cl(-) conductances after hypotonic cell swelling. Hence, H(2)O(2) induces KCl cotransport or K(+) and Cl(-) channels in nonadherent and adherent cells, respectively.
Assuntos
Carcinoma de Ehrlich/metabolismo , Adesão Celular , Canais de Cloreto/metabolismo , Fibroblastos/metabolismo , Estresse Oxidativo , Canais de Potássio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Simportadores/metabolismo , Animais , Ácidos Carboxílicos/farmacologia , Linhagem Celular Tumoral , Tamanho Celular , Inibidores Enzimáticos/farmacologia , Feminino , Peróxido de Hidrogênio/metabolismo , Soluções Hipotônicas , Indenos/farmacologia , Transporte de Íons , Toxinas Marinhas , Camundongos , Células NIH 3T3 , Nitratos/metabolismo , Pressão Osmótica , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Fatores de Tempo , Cotransportadores de K e Cl-RESUMO
The mechanism of activation of the transient receptor potential vanilloid 4 (TRPV4) channel by 4alpha-phorbol esters was investigated by combining information from chemical modification of 4alpha-phorbol-didecanoate (4alpha-PDD, 2a), site-directed mutagenesis, Ca(2+) imaging, and electrophysiology. Binding of 4alpha-phorbol esters occurs in a loop in the TM3-TM4 domain of TRPV4 that is analogous to the capsaicin binding site of TRPV1, and the ester decoration of ring C and the A,B ring junction are critical for activity. The lipophilic ester groups on ring C serve mainly as a steering element, affecting the orientation of the diterpenoid core into the ligand binding pocket, while the nature of the A,B ring junction plays an essential role in the Ca(2+)-dependence of the TRPV4 response. Taken together, our results show that 4alpha-phorbol is a useful template to investigate the molecular details of TRPV4 activation by small molecules and obtain information for the rational design of structurally simpler ligands for this ion channel.
Assuntos
Ésteres de Forbol/química , Ésteres de Forbol/farmacologia , Canais de Cátion TRPV/metabolismo , Acilação , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Esterificação , Humanos , Camundongos , Relação Estrutura-Atividade , Canais de Cátion TRPV/agonistasRESUMO
Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used immunofluorescence microscopy, Western blot analysis and alkali comet assays to show that phosphorylation of ATM in NIH3T3 fibroblasts occurs prior to apoptotic DNA fragmentation, nuclease degradation and phosphorylation of histone H2A.X in cells treated with low levels of either staurosporine (STS) or tumor necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated independently of DNA damage signaling pathways during the very early stages of apoptosis.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Caspase 3/metabolismo , Cromatina/patologia , Cicloeximida/farmacologia , Dano ao DNA/fisiologia , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática , Soluções Hipotônicas/farmacologia , Camundongos , Células NIH 3T3 , Fosforilação , Estaurosporina/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches, that three MAPKs exhibit unique interrelationships with the Na(+)/H(+) exchanger, NHE1, after osmotic cell shrinkage: Extracellular Signal Regulated Kinase (ERK1/2) is inhibited in an NHE1-dependent, pH(i)-independent manner, c-Jun N-terminal kinase (JNK1/2) is stimulated, in part through NHE1-mediated intracellular alkalinization, and p38 MAPK is activated in an NHE1-independent manner, and contributes to NHE1 activation and ERK inhibition. Shrinkage-induced ERK1/2 inhibition was attenuated in Ehrlich Lettre Ascites cells by NHE1 inhibitors (EIPA, cariporide) or removal of extracellular Na(+), and mimicked by human (h) NHE1 expression in cells lacking endogenous NHE1 activity. The effect of NHE1 on ERK1/2 was pH(i)-independent and upstream of MEK1/2. Shrinkage-activation of JNK1/2 was attenuated by EIPA, augmented by hNHE1 expression, and abolished in the presence of HCO(3)(-). Basal JNK activity was augmented at alkaline pH(i). Shrinkage-activation of p38 MAPK was NHE1-independent, and p38 MAPK inhibition (SB203580) attenuated NHE1 activation and ERK1/2 inhibition. Long-term shrinkage elicited caspase-3 activation and a loss of cell viability, which was augmented by ERK1/2 or JNK1/2 inhibition, and attenuated by p38 MAPK inhibition.